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Cell Biology Laboratory MCB 14OL Winter Quarter 2013 MOLECULAR AND CELLULAR BIOLOGY 140L WINTER QUARTER 2012 Course Description This is a ve unit lecture laboratory course that will teach students the techniques used by modern day cell biologists Our goal is to teach the students how each different technique can uniquely address cell biological problems If you have taken BIS104 you should recognize many of the techniques and it would be valuable to review your notes andfor text as our experiments unfold The simple eukaryote S cerevisiae is a budding yeast and will be our primary model system because of the ease with which genetic cytological biochemical and molecular genetics approaches can be applied Due to the heavy demand on lab space personnel and equipment there will be no opportunity to make up missed labs If one or two labs are missed due to a documented illness or preapproved absence you will be required to submit a labwrite up see Dr Kaplan for details one week after the missed lab Your grade will be determined based on labs completed and the quality of your lab writeups Lead Instructor Dr Ken Kaplan 204 Briggs Hall 7545044 kbkaplanucdavisedu Teaching Assistants Lab Section I Kari Messina klmessh1augdavisedu Lab Section 11 Michelle Panzica Roving TA Jonny Diehl jsdiehlucdavisedu Class Time Lecture T R 1100 1150 AM Hart Hall 1150 Discussion F 110 200 PM Hoagland Hall 113 Lab Section I T R 1210 300 PM SLB 3051 Lab Section II T R 1210 300 PM SLB Course Grade Percent of Grade EXAMS 2 40 LAB PRACTICAL 20 LABORATORY 15 WRITING ASSG 15 DISCUSSION 10 The laboratory component of your grade includes your record of attendance participation commitment and ability as well as laboratory notebook keeping and inclass quizzes LABILECTURE SCHEDULE Lecture I Date Lecture I Lab Topic D Lab 1 107 Introduction PippettingAcids and Bases Yeast 2 Hybrid Transformation 2 109 Protein concentration Bradford Assay 3 114 Cell Fractionation Membrane organelles 4 116 Cell Fractionation Protein Concentrations 5 01 21 I Membrane topologylassociation 6 123 SDSPAGE and Western Transferf Streak Transformants on Selection 1B in 7 128 Western Blot Detection and 2hybrid analysis IC 7 130 Western Blot analysis and 2hybrid model building 8 204 TAP af nity puri cation I Exam1 0206 TAP Af nity puri cation II SDS PAGEWestern Lab 10 10111 I P 211 I I Western Blot Detection and data Frxnmembrane topology analysis Light Microscopy 10111 213 Light Microscopy Western Blot Detection and data frxnmembrane topology A T analysis 1213 218 Fluorescent Microscopy I Septins in wild type cells Advanced uorescence microscopy 12 13 220 Advanced fluorescence microscopy Fluorescence Microscopy I Septins in wild type cells 1415 227 Septin and cytoskeleton indirect IF of mutants 39 Biological Databases 1415 304 2 Fluorescence microscopy II mutant analysis Septin and cytoskeleton indirect IF of mutants 15 16 306 X Biological Databases Fluorescence microscopy II mutant analysis 15b 311 0 IF analysis and gure making and microscopy review Exam 2 0313 Practical 3I7 Instruction ends Note exam is followed by lab on the same day Lab section I Lab section II Writing assignment rst draft is due 313 Your nal draft is due 3117 2 MCB l40L LABORATORY 1 PART 1 COURSE INTRODUCTION 1 Lab Safety Be aware of potential hazards at all times Your safety should of highest concern to you Follow these simple rules and ask questions if you are unsure No food or drink allowed in the lab area use the hallway to get a drink or enjoy a coffee Wear long pants that cover your knee when sitting and no opentoed shoes ll Wear protective eyeglasses when necessary Tie hair back especially when working with ames No mouth pipetting ever Broken glass needs to be disposed of in glass waste box In the event of a re alarm tum off all ames shut down equipment evacuate to area outside of SLB by the BioBrew do not leave until instructor has checked that all students are out Know where the re extinguishers eye washes and rst aid kits are positioned To use a re extinguisher pull out metal pin near the handle point nozzle at base of ame and squeeze handle Materials Safety Data Sheet MSDS can be accessed httprchemwatchnet chemwatchwebdashboard Personal protective wear We encourage you to use gloves and safety glasses when handling chemicals or biologicals in the lab especially if you are unsure about their relative risk askl In addition you will be instructed on which procedures REQUIRE gloves and safety glasses Lab coats are not required for the procedures we carry out but you are welcome to provide your own to wear 1 The Instructors and Staff involved in the MCB teaching labs whenever possible have eliminated or greatly reduced the use of hazardous and carcinogenic chemicals Also the protocols have been tailored to eliminate or greatly reduce hazards from spilling splashing breaking etc mostly by using small volumes and directing the use of pipets for acquiring solutions instead of pouring from large bottles 2 Use of Safety Glasses These are one size tsall so secure them as best you can They are impact resistant and block UV light You may wish to clean the glasses before use if so then please use the mild hand soap and tap water to gently clean the glasses and dry with a paper towel When nished using the glasses please return them Mandatory use of glasses i When viewing agarose gels with hand held UV lamps ii When heating samples in SDSsample buffer at 100 C iiiWhen melting agarose in the microwave 3 Use of Gloves There are small medium and large gloves available They are disposable so please throw them away as directed Also since the purpose of gloves is to prevent chemicals or bacteria from getting on your hands then please remove the gloves when nished Do not keep used gloves on to perform other tasks as this spreads the chemicals or bacterial Mandatory use of gloves i When handling acrylamide even using precast gels iiWhen handling agarose gels iiiWhen handling bacteria Details will be provided and discussed by the Instructor in charge and or in the lab manual 2 Lab Courtesy Never stick your personal pipettes in a community bottle and never put excess reagents back into community supplies Never take community reagents back to your own bench Don t use more chemicals than you need Always clean up after yourself Speci cally rinse and remove tape from glassware prior to putting into designated basins Also place broken glass and other toxic substances into designated containers 3 The Concept of Signi cant Figures Signi cant gures re ect the accuracy and precision of the instruments that make the measurements For example if a balance reads 1567 g there are four signi cant gures where the last gure is probably an estimate A cruder balance might read 16 g and there would only be two signi cant gures Familiarize yourself with the application of signi cant gures in multiplication and additionfsubtractionf Review the concepts of accuracy and precision 4 Concentration of Solutions wvpercent weight to volume grams100ml vv percent volume to volume ml100ml 5 Dilutions Simple way of doing dilution problems C1V1C2V2 Where C1 and V initial concentration and volume respectively And C2 and V2 target concentration and volume respectively Dilution factors nal volume divided by the initial volume C2 C1Dr Multiple or serial dilutions are sometimes the only way to dilute accurately because of the accuracy of measuring devices see below An alternative method using the dilution factor C2C1Df dilution factor Df V2V1 V 1 volume of stock solution to add 6 The proper usage of Graduated Cylinders Pipets and Pipetmen The appropriate volume ranges for each for maximum accuracy 4 The correct usage of blowout graduated glass pipets and bulbs The correct usage of pipetrnen and how to calibrate MCB l40L LABORATORY 1 PART 2 SOLUTIONS AND PIPETING One key component in any biochemicalcell biological assay is a buffer A buffer is generally a weak acid At low pH weak acids are protonated at high pH unprotonated Since the acquisition of a proton can cause an uncharged base to take on a positive charge i e NH3 H lt NH4quot39 or can neutralize a negative charge z39e RCOO39 H lt gt RCOOH the ionic forms of the many molecules that exist in a cell are very much dependent on the intracellular pH Biologically important weak acids have a wide range of acid dissociation constants This creates two challenges for the experimentalist The first is to determine which ionic fonns are most appropriate in a particular cellularexperimental setting The second is to set and maintain the pH at a value that will assure appropriate levels of the quotbiologically activequot ionic forms of a particular weak acid This second challenge also is faced by the cell as it conducts its normal functions in an environment where protons are being produced andfor consumed in a myriad of reactions The pH is set by xing the ratios of protonated and unprotonated forms of all ionizable groups within the solution The relation between pH and the ratios of the protonated and unprotonated forms of weak acids and bases is described by the HendersonHasselbalch equation The HendersonHasselbalch equation can be derived from the eqilibrium relationship between a weak acid and its conjugate base HA gt Hquot39 A391 and is the cell biologist s tool to creating a solution that will buffer in the desired pH range HendersonHasselbalch equation PH PKa39 103 AlHA In the following section you will be asked to create a virtual buffer Speci cally using the HendersonHasselbalch equation you will calculate the correct amount of buffer components to add to a solution Preparation of a Buffer Method 1 01 M TrisHCl Bu er pH 80 1 Determine the volume of 1 M Tris base pKa 821 at 25 C needed to prepare 100 ml of a 01 M Tris solution Write this volume below ml 1 M Tris base 2 Using the HendersonHasselbalch Equation and the pKa for Tris calculate the amount of 1 N HCl needed to adjust the pH to 8 Write this value below and record all of your calculations in your lab notebook ml 1 N HCl Calculated As a researcher you must be capable of creating complex solutions from stocks Below is an example of a typical complex buffered solution that we will use later on in the course to stabilize enriched mitochondria prepared from yeast cells 50 mM HEPES pH 74 06 M Sorbitol 1 mM PMSF HEPES is a weak acid that has been chosen as a buffer Sorbitol is an osmolyte that has been chosen to stabilize the mitochondrial membranes and PMSF is a serine protease inhibitor To create 200 mls of this complex solution a researcher could weigh out all three compounds and dissolve them together in 200 mls This approach is valid but lacks exibility To obtain maximum exibility researchers create stock solutions that are concentrated preparations of materials that comprise complex solutions In our case we have decided to create the following stock solutions 1 M HEPES pH 74 2 M Sorbitol 01 M PMSF Please indicate below the volume of each stock you would need to add in order to obtain 200 mls of the complex solution given in the table above Volume added 1 M HEPES pH 74 2 M Sorbitol 01 M PMSF Describe the steps you would take to prepare the solution Pipeting Please review before continuing onto Part 3 1 Adjust the pipetting device to 09 ml 900 pl 2 Attach a disposable tip to the end of the pipette shaft and press on rmly 3 Depress the plunger to the FIRST RESISTANCE POINT and hold in place 4 Holding the pipetter vertically immerse the end of the disposable tip into a beaker of water to a depth of 24 mm With the tip immersed in water gently release the plunger and allow the piette tip to ll with liquid Wait a few seconds to make sure that the full volume of liquid is drawn up into the pipette Note be sure that air bubbles are not drawn up into the pipette 5 Withdraw tip from the water and make sure that no liquid remains adhered to the outside of the pipette tip 6 To dispense sample place the tip against the side wall of a 15 ml plastic microcentrifuge tube in a tube rack and depress the plunger slowly to the FIRST POSITIVE STOP and then continue depressing the plunger through to the SECOND FULL STOP position to fully eject the liquid in the pipette tip Note placing the pipet tip in contact with the plastic surface of the microcentrifuge tube is critical to reproducible pipetting The contact tends to leave about the same amount of liquid on the pipet tip at each stage so that the volume transferred is reproducible 7 With the plunger still fully depressed withdraw the pipette from the microcentrifuge tube 8 Allow plunger to slowly return to the TOP POSITION prior to pipetting another sample or discarding the used pipette tip 9 Repeat this process until you can do it easily MCB 140L LABORATORY lA PART 3 PROTEIN PROTEIN INTERACTIONS YEAST TWOHYBRID Transform yeast with AD activation domain and BD binding domain vectors For this experiment we will NOT be working in pairs Each student will be responsible for conducting the following 1 Grow yeast to log phase TA will provide these cells Find out the number of cellsml in the culture so you can calculate the number of cells usedtransformation and the efficiency of transformation co1oniestotal cells x 100 2 Pellet 12 mls of cells by placing them in a 15 ml conical tube and centrifuging in a clinical centrifuge at maximum RPMS for 5 minutes decant supernatant 3 Wash with 10 mls of sterile H20 by resuspending the cell pellet in sterile H20 using a sterile 10 ml pipet or a sterile tip on a P1000 pipet1nan pellet cells again by centrifugation and remove supernatant carefully by decanting 4 Resuspend cell pellet in 310 ul of 100 mM LiOAc2 5 Aliquot 50 ul to six 15 ml sggrilg eppendorf tubes labeled 16 see table below Be sure to also provide your initials or other mark on the tubes so you can identify them everyone s tubes are together 6 Pellet cells by centrifugation in a microfuge at maximum RPMS for 1 minute remove supematant using a pipetman with a sterile tip 7 Add the following reagents to each tube containing a cell pellet 240 ul 50 PEG 2000 36 ul 1M LiOAc2 70 ul sterile dH2O 5 ul boiled ssDNA 1 ul of each plasmid as designated in Table below 8 Mix well by pipeting up and down using a sterile tip 9 Incubate 30 C for 30 minutes in a temperature block 10 Incubate at 42 for 15 minutes in a temperature block 11 Plate entire reaction onto Leu Ura media in plates using sterile glass beads see TA s for demonstration 12 Place Leu Ura plates at 30 Colonies should appear after 23 days PLASMID Tube Label Activating Domain Vector Binding Domain Vector 1 BD Cdcl 1AD 2 Cdcl 1BD Cdc11347 415 AD 3 Cdcll101217BD CdcllBD 4 Cdc12BD Cdc11A347 415AD 5 Cdc3 BD Cdc11A347 415AD 6 Cdc3 BD Cdcl 1AD 8 MCB l40L LABORATORY 2 BRADFORD PROTEIN ASSAY Theoretical Basis for Spectrophotometric Analyses Relation of Absorbance to Concentration The absorbance A of a light absorbing solution also called optical density OD is de ned by Beer39s Law A log 101 1 where I0 is the intensity of the incident monochromatic light and It is the residual intensity after passing through an absorbing solution Ideally absorbance depends on the length of the optical path 1 and the concentration of the absorbing material c according to the equation Aelc 2 s is a constant that is characteristic of a lightabsorbing species at a particular wavelength When 1 is expressed in cm and c in molesliter i e M s has the dimensions M 1cm391 and is called the molar absorptivity or molar extinction coef cient The molar absorbance of a compound at a given wavelength is the absorbance of a 1 M solution of the compound when the light path is 1 cm The values of A for single absorbing groups tend to fall in the range of 102 9 105 M 1 cm391 Since an absorbance of 02 can be measured with good precision approximately 1 x IO 6 M solutions of strongly absorbing substances can be analyzed in a 1 cm cuvette see Freifelder page 497 for a more complete discussion The molar absorptivity varies with changes in temperature pH ionic strength and type of solvent as well as the wavelength of the incident light 10 The light path 1 for a given instrument and cuvette is generally known Thus the concentration of a solution of a absorbing substance can be calculated from a single absorbance measurement and knowledge of the 2 value This is apparent from the simple rearrangement of equation 2 to yield c As 1 3 or c Me for a 1 cm light path 4 Equation 2 is more convenient to use in analytical work than is equation 1 It states that the absorbance is directly proportional to the concentration of the absorbing compound It must be noted however that this simple relation may not hold in concentrated solutions Because of this the range of concentration with linear relationship to absorbance must be determined empirically for each substance Solutions to be subjected to spectrophotometric analysis are generally examined after being placed in special vessels called cuvettes The absorption properties of the materials used in the construction of cuvettes determine the usefulness of these vessels for spectrophotometric work i e the cuvettes themselves can interfere Inexpensive plastic cuvettes work well in the 9 visible range of the electromagnetic spectrum gt 300 nm Below 300 nm these cuvettes are excellent absorbers themselves Special glass cuvettes may be used for spectral work down to 240 nm Below 240 nm quartz cuvettes must be used High quality doublebeam spectrophotometers such as the Shimadzu used in our laboratory employ rectangular 1 cm light path cuvettes for measuring the absorbance of solutions With a Shimadzu spectrophotometer reliable readings can be made up to an absorbance of 20 Above 20 measurements become inaccurate The Shimadzu reports all absorbances over 25 as 25 Therefore any reading of 25 has no meaning In every spectrophotometric analysis it is necessary to have a reference or blank solution in addition to the standards and unknown solutions The blank solution is used to eliminate effects of solvent absorbance The blank solution may contain only the solvent in which the lightabsorbing compound is dissolved but preferably also contains all the reagents used in the determination reagent blank A careful experimentalist records the absorbance of the blank and subtracts this value from the absorbance of each sample Quantitative analyses are an essential part of almost every investigation in cell biology In some cases chemicalbased assays are required because the substances to be measured are present in milligram microgram or even nanogram amountswell below the detection limit of classical gravimetric and volumetric procedures Because these compounds are usually present in complex mixtures such as cellular extracts assays must be selective measuring only the compound of interest Experiment 2 provides experience in following a simple protocol for spectrophotometric analyses of protein concentration by the Coomassie blue dyebinding assay A protocol is a detailed set of instructions on how to perform an experiment usually in outline form There are several spectrophotometric assays for protein concentration that differ in sensitivity speci city and convenience This experiment will introduce the Bradford method A rapid and sensitive method for quantitation of protein in solutions was developed by Bradford 1976 and later improved by Read and Northcote 1981 This method depends upon the shift in absorbance maximum max of the dye Coomassie Brilliant Blue G250 from 465 nm to 595 nm caused by the binding of the dye to the hydrophobic regions in a protein The solution visibly changes from brown to blue as the dye binds to protein The increase in absorption at 595 nm is monitored and can be linearly related to protein concentration under ideal conditions The reaction is complete within two minutes the color is stable for about one hour Depending on the protein the assay is useful up to 125 pg of protein in the assay volumes usually used Detergents such as sodium dodecylsulfate SDS or Triton X100 interfere greatly with the dyebinding assay Smaller effects are caused by Tris buffer mercaptoethanol sucrose glycerol or ethylenediaminetetraacetic acid EDTA The structures of some of these compounds are given in Figure 1 TC Coomassie blue Hac S quot mercaptoethanol H20quot so Figure 1 Structures of Coomassie Blue G 250 SDS and mercaptoethanol Interfering Substances Selectivity is always of prime importance in judging the merits of a particular assay During the isolation of an enzyme the biochemist must measure the protein content of complex solutions containing the multitude of substances present in the crude cell extract plus the buffers salts and other compounds that are added during the isolation The ideal protein analysis would be sensitive only to total protein and not be in uenced or interfered with by other substances The ability to compensate for the presence of an interfering substance depends on its mode of action The substance may react with the reagents and cause an absorbance increase when added to a reagent blank For example an A595 of 0105 might be observed when 200 pg of interfering substance X is added to the blank in the Bradford assay From examining a standard curve for the assay we might determine that the absorbance due to 200 ng of X is equivalent to approximately 2 ng of protein Knowing this relationship between the A595 arising from given amounts of X and of protein allows the experimenter to estimate possible errors due to X and to predict whether the effects of a xed amount of X in the protein solution can be compensated for by including X in a reagent blank as a control If however X causes a very large increase in the absorbance when mixed with the reagents the assay will not be workable because the total absorption could exceed the range of the spectrophotometer or the reagents could be insuf cient to react with both X and the protein Another type of interfering substance may affect the ability of proteins to react with the reagents It would have no effect on absorbance when added to a blank in contrast to the above paragraph but nevertheless cause either a decrease or an increase in the assay absorbance when protein is present Therefore it is necessary to test potentially interfering substances both in the absence and presence of the compound being assayed Figure 2 is an example of a standard absorbance curve from a protein assay To construct such a standard curve a series of protein solutions with a range of known concentrations is 11 9 prepared These are all measured under identical conditions and plotted as absorbance vs concentration To use the standard curve the measured absorbance of an unknown sample examined under the same conditions is located on the standard curve and the corresponding concentration determined on the abscissa According to Figure 2 at low concentrations of this protein the assay obeys the proportionality described by Beer39s Law ie it gives a linear standard curve At higher protein concentrations the curve becomes nonlinear This failure to obey Beer39s Law at higher protein concentrations can be of chemical or physical origin Incomplete formation of a colored product to be analyzed absorption by interfering substances and selfassociation are examples of sources of nonlinear responses An assay in which Beer39s Law is not obeyed can still be made useful by measuring the absorbance at many known standard concentrations as in Figure 2 and interpolating between known concentrations By taking many points the curve is more precisely de ned in the nonlinear regions present in solutions containing unknown amounts of test compound Standard Curve 08 07 PF 06 05 o4 Absorbance 03 02 01 0 1 2 3 4 5 6 7 8 9 10 1 1 ug Proteinmssay Volume Figure 2 Standard curve for a quottypicalquot protein assay References Colorimetric Determinations of Proteins 1 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Analytical Biochemistry 1976 May 7 72224854 2 Read SM and Northcote DH Minimization of variation in the response to different proteins of the Coomassie blue G dyebinding assay for protein Analytical Biochemistry 1981 Sep 1 116l53 64 12 MCB 140L PROTOCOL 2 BRADFORD ASSAY Quantitative protein analysis by Coomassie blue dyebinding Standard Curve Construction and Determination of the Concentration of an Unknown Protein See Protocol in Table 1 1 Label 26 microcentrifuge tubes with the numbers 1 to 26 with a Sharpie 2 Pipette the desired amount of deionized water into the tubes according to the amounts shown in Table 1 3 Prepare the following stock solutions 0100 ugml BSA The BSA is supplied at a concentration of 20 mgml Prepare 15 ml of this dilution in water in a 15 ml microcentrifuge tube Make sure this solution is well mixed 0100 ugml Lysozyme Prepare 200 pl from a 2 mgml solution o100 pigml Gelatin Prepare 200 iii from a 2 mgml solution 4 Add the appropriate amount of the protein stock solutions unknown protein andor additive to each tube Note these solutions have to be prepared rst Tubes 114 represent the protein standard curve in duplicate using bovine serum albumin as a protein standard A standard curve is always prepared at the same time that you are determining the concentration of an unknown protein tubes 1520 Tubes 2126 contain other proteins for comparative purposes andor substances which potentially interfere with the Coomassie blue assay 5 When all of the protein solutions have been prepared add the Bradford reagent and mix immediately by inverting the tube twice 6 Transfer 200 ul of each solution to a well in a 96 well plate Record which well contains each sample in your lab book Measure the absorbance at 595 nm 7 Write the measured values in Table 1 Subtract the OD595 value for the blank from your protein samples Construct the protein standard curve by plotting the measured absorbance versus protein amount in ng Calculate the protein concentration ugl ml of the unknown using the average value of duplicate dilutions Sensitivity and Interference Since the amount of an unknown protein is to be estimated using the BSA standard curve it is important to know the reactivity of the Coomassie blue assay with different proteins The relative sensitivities of assays for BSA lysozyme and gelatin can be calculated by comparing tubes 21 and 22 to the BSA standard curve Tubes 23gt26 test three nonprotein potentially interfering substances for their ability to affect the A595 in the presence and absence of protein These results will provide measures of the selectivity of the Coomassie blue reagent ie its ability to give A595 values that depend on the protein concentration and to discriminate against the potentially interfering substances After performing these experiments answer the following questions 1 Which if any of the potentially interfering substances complex buffer and SDS screened actually interferes If there is one give the chemical basis for its interference If a known small amount of the substance was present in an assay tube could you compensate for its effect Explain 13 2 Calculate the sensitivity percent of maximal color reaction of this assay towards the three different proteins tested BSA lysozyme and gelatin If there is a difference in sensitivity give an explanation for why Table 1 Protocol for Bradford protein assay Tube Volume of Standard or Water Reagent Total Protein A595 A595 Sample in tube avg 1 0 ul of 100 Lgml BSA 500 pl 500 ptl 0 pg 2 0 ptl of 100 ptgml BSA 500 pl 500 ul 0 pig 3 15 ptl of 100 pugml BSA 485 pl 500 311 15 ug 4 15 pl of 100 pigml BSA 485 pm 500 ptl 15 pg 5 30 ul of 100 pugml BSA 470 pl 500 pl 30 pg 6 30 ptl of 100 ugml BSA 470 pl 500 pl 30 pg 7 45 ul of 100 ugml BSA 455 pl 500 pl 45 pig 8 45 ptl of 100 ugml BSA 455 pl 500 pl 45 pg 9 60 ptl of 100 pgml BSA 440 pl 500 111 60 pg 10 60 ul of 100 ugml BSA 440 ul 500 pl 60 pg 11 75 ul of 100 ugml BSA 425 ul 500 111 75 pig 12 75 ul of 100 pigml BSA 425 ul 500 M1 75 pig 13 90 ul of 100 ptgml BSA 410 ul 500 pl 90 pig 14 90 ul of 100 pigml BSA 410 ul 500 pl 90 ug 15 500 pt unknown 110 0 ptl 500 ul ie 50111 in 45014 16 quot quot 110 quot 500 ul 17 quot quot 150 quot 500 iii 18 quot quot 150 quot 500 ul 19 quot quot 1100 quot 500 ul 20 quot quot 1100 quot 500 ul 21 60 pl 100 ugml lysozyme 440 ii 500 pl 60 pg 22 60 pl 100 ugml gelatin 440 pl 500 JJ 60 pg 23 25 ul 06 M sorbitol 50 mM 475 pl 500 ill 0 ug HEPES 1mM PMSF 24 25 ul 06 M sorbitol 50 415 pl 500 tr 60 pg mM HEPES 1mM PMSF 60 ul 100 pigml BSA 25 25 pl 4 mgml SDS 475 pl 500 pl 0 pg 26 25 M14 mgml SDS 415 ptl 500 ul 60 pg 60 pl 100 pgml BSA Final unknown ugml I4 PLOT 5f390UI CUR T5 MCB 140L LABORATORY 3 Cell FractionationLysis and Differential Centrifugation Introduction A host of fractionation procedures are employed by cell biologists Each organelle has characteristics size shape and density for example that make it different from other organelles within the same cell If the cell is broken open in a gentle manner each of its organelles can be subsequently isolated Cell Lysis In order to isolate and analyze subcellular organelles cells must be lysed or broken open In many cell types such as human cells the plasma membrane can be directly ruptured using a variety of force generating techniques that depend on ripping or shearing cells chemical treatments such as detergents or osmotic disruption For budding yeast cells however the hard cell wall must rst be removed enzymatically using an enzyme called Lyticase or zymolyase The yeast cells you will be given in lab will have already been treated with this enzyme to remove the cell wall This will cause the cells now referred to as spheroplasts to be very osmotically fragile and prior to lysis they must be treated gently ie no vortexing or harsh pipeting You will lyse the spheroplasts by bead beating using glass beads and a vortex Bead beating uses many small beads to shear off the plasma membrane Other methods such as homogenization force cells through a small space that lies between the pestle and the glass container thereby generating shearing forces that cause the plasma membrane to rupture Care must be taken during lysis to keep the cell extract as cold as possible to avoid proteolysis and protein denaturation Once the cells are lysed this is called your total cell extract What is in this Cell Extract Unbroken cells organelles such as nuclei mitochondria golgi vacuoles and cytosol Your goal is to fractionate this total extract to obtain fractions that are enriched for mitochondria and cellular membranes i e microsomes that include but are not limited to plasma membranes Fractionation One of the most commonly employed techniques for the fractionation of cellular organelles is centrifugation Centrifugation is simply the application of radial acceleration to extract particles by rotational motion Intuitively particles that are denser than the medium will sediment and particles that are lighter will oat In a cell extract this means that organelles will pellet upon centrifugation with the exception of fat globules which because of the buoyant nature of lipids will oat to the top after centrifugation Centrifugation requires a rotor rotor tubes and a centrifuge There are many different types of rotors such as xed angle swinging bucket or vertical and their different names re ect how the rotor tubes are placed within them and the resulting centrifugal force the samples experience There are also different types of centrifuges such as a table top or clinical and microfuge that you have already used in your other experiments In addition there are superspeed and ultracentrifuges Different centrifuges are capable of rotating rotors at different speeds with the 16 utltracentrifuge capable of the fastest We will utilize superspeed centrifuges to fractionate our yeast extracts There are basically three centrifugation techniques employed by cell biologists to fractionate extracts velocity sedimentation sedimentation equilibrium and rate zonal separation Velocity sedimentation is the technique we will employ to fractionate our yeast extracts and it depends on the size of the particle the shape of the particle and the viscosity of the media These three variables are described by the sedimentation coef cient S value or Svedburg in 10 I3 sec of a given particle The table below gives some known S values of biological particles S value 10 13 sec Type of particle 015 Proteinsprotein complexes 40400 Ribosomal subunitspolysomes 40000 mitochondria 100000 golgi ER and plasma membranes microsomes Differential centrifugation is an application of velocity sedimentation that we will perform This technique simply utilizes increasing forces andor longer centrifugation times to successively pellet different particles within a given extract Here is a helpful website that discusses the theory of sedimentation httpbrickertcnjedutech BIOL3 1 1centrifugationhtml Read the protocol that we will employ to lyse and fractionate yeast extract to isolate mitochondria microsomes and proteins To aid in understanding the protocol in the space provided construct a ow chart of the steps that we will use to fractionate our cell lysate Complete this before coming to class 1 MCB 140L Protocol 3 Cell Fractionation Enrichment of Mitochondria and Cellular Membranes All bu er components are listed at the end of the protocol and will be prepared for you Read and prepare in advance This experiment will be performed in groups of two TA s will complete before lab I Grow yeast cells to n1idlog phase Please nd out the concentration of yeast and the exact amount of material you received so you can calculate the of total cells you used 2 Harvest cells by centrifuging at 2700 rpm SLCS6000 rotor for 5 minutes 3 Resuspend the cell pellet in 50 mls of dH20 and harvest cells by centrifuging at 3500 rpm SLCS6000 rotor for 5 minutes 4 Resuspend in 8mlsL of culture in NMIB and transfer to a 15 ml conical tube Pellet in table top centrifuge setting 7 for 5 minutes 5 Resuspend pellet in volume of NMIB 1X protease inhibitors lmM PMSF 5mM benzamidine equal to pellet volume 2mlsL 6 Freeze cells by drop method in liquid N2 and store at 80oC until time to lyse 7 Lyse cells in freezer mill Lysates were aliquoted and 05 ml were frozen in liquid N2 until fractionation To be completed in lab on ice 8 Thaw 05 ml lysate and add protease inhibitors lmM PMSF and 5mM benzamidine 9 Mix in eppendorf tube and spin 3000 X g 2500 rpm in coldroom microfuge for 5 minutes 10 SAVE supernatant which contains mitochondria transfer to clean 15ml eppendorf centrifuge tube What is in this pellet Transfer 25 ul of this supernatant to a new eppendorf tube and label it TOTAL 11Spin rest of supernatant at 14800 x g 13200 rpm in coldroom microfuge for 15 min Save supematant label PMS for PostMitochondrial Supematant l2Resuspend pellet in 50 ul of NMIB and transfer to eppendorf tube labeled MITO 13 Aliquot 10 ul of MITO and 10 ul of PMS fractions into separate newly labeled eppendorf tubes to be used for Bradford Assay during next class period 14 Spin remaining PMS at 80000 rpm 250000 x g for 10 in a TLA100 rotor in the table top ultracentrifuge the TA will help you load the centrifuge and multiple groups should spin together to save time 15 Resuspend the pellet in 25ul NMIB and label HSM high speed membrane pellet 16 Save the supernatant and label HSS high speed supernatant 17 Aliquot 25ul of HSS to a separate newly labeled eppendorf tube to be used for Bradford Assay during the next class period 18 Give all fractions and aliquots ve labeled tubes for one actionation to instructors to freeze in liquid nitrogen NMIB 06 M sorbitol 20 mM HepesKOH pH 74 18 5mM Magnesitun chloride 50mM Potassium chloride 100mM Potassium acetate 1 mM PMSF 5 mM benzarnidine add protease inhibitors just before use inactivated quickly upon dilution MURB 100 mM MES pH 70 1 SDS 3 M Urea 5 betaMercaptoethanol 19 Flow chart of yeast lysate fractionation 20 MCB 140L LABORATORY 4 Cell FractionationProtein Assay We will use SDSpolyacrylamide gel electrophoresis PAGE and Western blot to assess the success of our yeast extract fractionation experiment In addition we will also analyze the association of septins with cellular membranes using an epitope tagged version of Cdcll Unlike transmembrane proteins septins have been suggested to interact peripherally with membranes through a block of basic amino acids However it remains untested what percentage of septins are membrane associated and how tightly they associate with membranes In order to analyze our fractions by SDSPAGE and Westem blot we rst need to determine the protein concentrations of the various fractions from Tuesday s experiment To determine protein concentration we will perform a Bradford Assay as we did earlier in the course Below is a protocol that you will use to generate a standard curve to estimate the protein concentrations in your various fractions Based on the estimated protein concentration of your various fractions shown below you need to determine how much volume of a given fraction and the dilution of that fraction you will need to assay to obtain an accurate estimation of the protein concentration of your samples using your standard curve PREPARE in advance by doing the calculations and reviewing the Bradford Assay from Lab 2 Fraction Estimated Protein Concentration mgml Total 5080 PMS 6080 MITO 2540 HSM 2040 HSS 5070 These numbers are estimates Please use them and the methods reviewed in lecture notes to practice calculations The estimates will be adjusted after the TAs have estimated the amount in their preps Calculations 21 Tube Volume of Standard or Water Reagent Total A595 A595 Sample Protein avg 1 0 pl of 100 pgml BSA 500 pl 500 pl 0 pg 2 0 pl of 100 pgml BSA 500 pl 500 pl 0 pg 3 15 pl of 100 pgml BSA 485 pl 500 pl 15 pg 4 15 pl of 100 pgml BSA 485 pl 500 pl 15 pg 5 30 pl of 100 pgml BSA 470 pl 500 pl 30 pg 6 30 pl of 100 pglml BSA 470 pl 500 pl 30 pg 7 45 pl of 100 pgml BSA 455 pl 500 pl 45 pg 8 45 pl of 100 pgml BSA 455 pl 500 pl 45 pg 9 60 pl of 100 pgml BSA 440 pl 500 pl 60 pg 10 60 pl of 100 pgml BSA 440 pl 500 pl 60 pg 11 75 pl of 100 pgml BSA 425 pl 500 pl 75 pg 12 75 pl of 100 pgml BSA 425 pl 500 pl 75 pg 13 90 pl of 100 pgml BSA 410 pl 500 pl 90 pg 14 90 pl of 100 pglml BSA 410 pl 500 pl 90 pg Sample Dilution Factor 15 500 pl Total 0 ul 500 pl 16 500 ul Total 0 ul 500 pl 17 500 pl Total 0 ul 500 pl 18 500 ul Total 0 ul 500 pl 19 500 pl Total 0 ul 500 pl 20 500 ul Total 0 ul 500 pl 21 500 ul Mito 0 ul 500 pl 22 500 ul Mito 0 ul 500 pl 23 500 ul Mito 0 ul 500 pl 24 500 ul Mito 0 ul 500 pl 25 500 ul Mito 0 ul 500 pl 26 500 ul Mito 0 ul 500 pl 27 500 ul PMS 0 ul 500 pl 28 500 ul PMS 0 ul 500 pl 29 500 ul PMS 0 ul 500 pl 30 500 ul PMS 0 ul 500 pl 31 500 ul PMS 0 ul 500 pl 32 500 ul PMS 0 ul 500 pl Set up two dilutions in duplicate for each of your samples Pilot P m s1anda1rd on L on the below 23 MCB 140L LABORATORY 5 Protein LocalizationProtease Protection Assay In these experiments we will assess the fractionation of total yeast extract by differential centrifugation using SDSPAGE and Western blotting with known markers that identify the mitochondria plasma membrane and cytosolic markers We will determine the nature of the interaction of septins with cellular membranes either using protease protection or ionic treatment of membranes and SDS PAGEWestem blotting Both of these biochemical techniques are focused on providing the cell biologist with information conceming the sub cellular localization of a given component The success of any given fractionation scheme can be evaluated by determining and comparing the speci c activity units of a given activitymg protein of various fractions collected during the puri cation In our case we have collected fractions following differential centrifugation representing total yeast extract postmitochondrial supernate fraction PMS a mitochondrial enriched pellet fraction mito a high speed membrane pellet HSP and a high speed supematant HSS To assess whether we obtained enrichment of mitochondria and cellular membranes using differential centrifugation we will load a constant amount of protein from each fraction and analyze them by SDSPAGE and Western blotting using antibodies directed against known cytosolic plasma membrane and mitochondrial proteins In order to load a constant amount of protein for analysis on SDSPAGE you may need to concentrate protein from each of your fractions using a precipitation technique If this is the case you will utilize trichloroacetic acid to denature your protein Denatured protein is insoluble and will form a precipitate that is easily collected by centrifugation in a microfuge Protease protection analysis is commonly used to assess the localization of proteins associated with sub cellular organelles Speci cally protease protection analysis is used to detennine whether a given protein is outside or inside a membranebounded organelle Proteins inside an organelle are protected from proteolysis by the lipid bilayer whereas those outside are fully accessible to exogenously added proteases and will be digested Proteins that interact with cellular membranes through one or many transmembrane domains are said to be integral membrane proteins Biochemically these proteins are de ned by their ability to associate with membranes under a wide range of pH and ionic conditions only detergent can release them from their interaction with membrane In contrast lumenal or peripheral membrane proteins can be dissociated from membranes under high pH or ionic strength buffers respectively We will use these conditions to investigate the nature of the interaction between septins and cellular membranes The yeast cells that you have fractionated contain a form of Cdc11 one member of the septin family expressed in yeast that has been genetically engineered to contain an tandemaf nity puri cation TAP tag The TAP tag is a form of epitope tagging Epitope tagging is a powerful and versatile strategy for detecting and purifying proteins expressed by cloned genes To utilize this feature proteins are typically engineered with a nucleotide sequence that encodes the peptide epitope tag or in this case multiple epitope tags fused together The gene of interest is cloned in frame relative to the tag and upon expression the protein of interest is synthesized as a fusion protein with the peptide tag Fusion protein detection andor puri cation is made possible by using highly speci c antibodies to the engineered peptide thus eliminating the need for 1 antibodies to proteins from each newly cloned gene Commonly used epitope tags include glutathioneStransferase GST c myc 6histidine 6XHis FLAG green uorescent protein GFP maltose binding protein MBP and in uenza A virus haemagglutinin HA The TAP tag we are using contains a protein A peptide that interacts with IgG constant region and a calmodulin binding peptide Consider how these two tags might be used together to purify the Cdcll protein from your extracts Please read the protocols below carefully before undertaking the experiment Any questions that you have should be asked before you get underway Have fun MCB 140L Protocol 5 Protease Protection Assay andor Salt Extraction Protease Protection Assay 1 Each Group Pair should obtain 1 tube of aliquoted microsomes that include secretory membranes such as but not limited to plasma membranes prepared by the TAs and identical to the HSP fraction you isolated previously 2 To wash the microsomes centrifuge at 4 C for 5 minutes at 80000 rpm in the TLA100 using the table top ultracentrifuge 3 CAREFULLY remove supematant using a pipetman 4 Remove supematant and resuspend in enough ul of MIB to yield a nal concentration of 4 mgml protein For example if you have 300ug total protein then resuspend in 75ul You will need to use your calculations of protein concentration for your fractions from last week 5 Set up 3 labeled tubes with the following conditions 1 100 ug of microsomes 2 100 ug of microsomes 100 ugml of proteinase K 3 100 ug of microsomes 100 ugml of proteinase K 01 Triton X100 Each reaction uses 25ul of microsomes Using the following stocks calculate the amount of each you will need and ll out the table given below BEFORE CLASS Stocks 4 mgml protein HSP fraction from steps 15 above 10 mglml Proteinase K 10 Triton X100 Bring up to 100ul with MIB Tube Microsomes HSP Proteinase K ul Trition X100 ul MIB ul ul 1 1 Place reaction tubes on ice for 30 minutes During this incubation set up your TOTAL MITO PMS HSP and HSS fractions for TCA precipitation as detailed below The TCA precipitations can be done in parallel so please read ahead and develop a ow chart to make these steps ef cient 2 After the 30 min incubation add 1 ul of 100 mM PMSF to tubes labeled 1 2 and 3 to inhibit the protease and mix well 3 Centrifuge as in step 2 above For tube 3 add 100 pl of 30 TCA generally used at 15 nal vv and go to step 6 why is this tube treated differently 4 Remove supernatant and resuspend pellet in a premixed solution containing 50 pl 30 TCA and 50 pl dH20 5 Mix well by vortexing the tubes 6 Place at 60 C for 5 minutes in a temp block located in lab 3 7 Place on ice for 10 minutes 8 Centrifuge all 6 tubes at 4 C for 10 minutes on high include Mitoprep fractions 9 Remove supernatant and resuspend pellet in 100 ul of acetone The pellet will not completely go into solution but you need to make sure it gets broken up Why is this step important 10 Centrifuge at 4 C for 5 minutes on high 11 Remove supernatant and allow pellets to air dry by leaving tubes open on ice for 10 minutes 12 Resuspend pellets in tubes 13 in 25 ul of MURB TCA Precipitation of fractions TOTAL MITO PMS HSM and HSS fractions Calculate how many microliters of each of your fractions you need to obtain 80ug of total protein If you don t have enough of your fraction for 80 ug use your all of your TOTAL and PMS MITO HSP and HSS IMPORTANT THE SDSPAGE GELS ONLY HOLD 2530ul OF LIQUID SO MAKE SURE YOUR FINAL VOLUME IS BELOW 25ul If the volume to be loaded on the SDSPAGE exceeds 25ul you will need to precipitate your fraction After calculating the volume of each fraction required for 80ug protein add MIB to a nal volume of 100ul Precipitate your protein by adding an equal volume ie 100ul of 30 TCA to each tube Mix well by inverting and vortexing and place on ice until you have reached Step 13 in the protocol above Make sure that your fractions have been on ice for at least 10 minutes for before proceeding Process your fractions as described above in steps 14 l8 Place all of your tubes containing MURB in a test tube rack and given them to the TAs for safekeeping Please label with your group and name Alternative assay Salt extraction of microsomes 1 Each Group Pair should obtain 1 tube of aliquoted microsomes that include secretory membranes such as but not limited to plasma membranes prepared by the TAs and identical to the HSP fraction you isolated previously 2 To wash the microsomes centrifuge at 4 C for 5 minutes at 80000 rpm in the TLA100 using the table top ultracentrifuge 3 CAREFULLY remove supematant using a pipetman 4 Remove supematant and resuspend in enough ul of MIB to yield a nal concentration of 4 mgfml protein For example if you have 300ug total protein then resuspend in 75ul You will need to use your calculations of protein concentration for your fractions from last week 5 Set up 3 labeled tubes with the following conditions Each reaction uses 25ul of microsomes 1 100 ug of microsomes 2 100 ug of microsomes NaCl to 025M SM stock 3 100 ug of microsomes NaCl to 05M Place membranes on ice for 30 minutes Centrifuge at 4 C for 5 minutes at 80000 rpm in the TLA100 using the table top ultracentrifuge 8 Carefully collect supematants and mark NaClSES concentration of salt extraction supernatant 9 Solubilize remaining membranes in 20ul MURB and label each NaClSEP 10 Add equal volume 2530ul of 30 TCA to each of the SES tubes and process as in steps 512 lO MCB 140L LABORATORY 6 PART 1 PROTEIN LOCALIZATIONSDSPAGE Today you will analyze your fractions from differential centrifugation and protease protection analysis on SDSPAGE and transfer the proteins separated by SDSPAGE onto membranes for Western Blotting analysis in the following laboratory Below is an excellent explanation of the theory behind SDS PAGE excerpted from the 120L laboratory manual Electrophoresis of Proteins in Polyacrylamide Gels Introduction All proteins possess charged amino acid side chains on their surfaces and hence are charged at some pH The net charge on a protein determines many of its physical properties such as its migration in an electric eld during electrophoresis or separation by ion exchange chromatography Polyacrylamide gel electrophoresis PAGE will be employed in this experiment to separate subunits dissociated by SDS denaturation on the basis of their size A special feature the electrophoretic system used in this experiment is the discontinuous buffer systems which lead to a higher resolution of the components in a protein mixture A rough estimation of the net charge on a protein can be obtained from the amino acid composition and appropriate pKa values However some amino acid residues such as tyrosine are buried in the nonpolar interior of a protein molecule do not contribute to the surface charge of the undenatured protein pKa values for various amino acid side chains have been measured by titration of proteins and model compounds A range of values is found for each amino acid see Table 1 because their pKas are in uenced by charged groups in close proximity in the folded polypeptide Table 1 pKa values of side chains of some amino acids in proteins Amino Acid pKa Value arg gt12 lys 97107 cys 8595 tyr 8510 his 6070 asp glu 3950 Theory of Gel Electrophoresis The rate at which a protein moves in an electric eld is de ned as its electrophoretic mobility Mobility is determined by a balance between forces imposed on the protein by the electric eld and the frictional drag on the protein It is a function of protein charge molecular weight and shape and of the particular experimental conditions used including eld strength voltage pH and ionic strength Positively charged proteins will migrate towards the cathode 5 negative electrode and have positive mobilities Negatively charged proteins will move towards the anode positive electrode and have negative mobilities Electrophoretic techniques follow migration of molecules in supporting media rather than free solution because superior resolution is achieved by minimizing convection and diffusion The supporting material is usually a gel composed of starch agarose or polyacrylamide Starch and agarose fomi noncovalent associations while acrylamide gels are formed by covalent cross1inking Polymerization of polyacrylamide gels proceeds by way of a chain reaction The rst step in gel formation is the activation of tetramethylethylenediamine TEMED by ammonium persulfate which leaves the TEMED molecule with a reactive unpaired electron The TEMED can combine with acrylamide which is thereby activated in tum to form polymers As the chain of acrylamide units grows the free radical is constantly regenerated at one end Bisacrylamide which consists of two acrylamide units joined through their CONH2 groups can be incorporated into two growing chains Hence the presence of bisacrylamide leads to the formation of crosslinks between chains With an abundance of crosslinks the polymer of a gel has a topologically complex conformation with loops branches and interconnections A possible experimental arrangement for the analysis of negatively charged anionic proteins by gel electrophoresis is shown in Figure 1 For positively charged cationic proteins the electrodes would be reversed The upper and lower reservoirs contain the same buffer that was incorporated into the gel though possibly at a lower concentration The buffer in the running gel serves not only to maintain a relatively constant pH but also as a conducting electrolyte Protein sample Upper buffer 39 resewoir Power Supply 4 Gel plates containing the polyacrylamide E Lower bu er El reservoir Figure 1 Polyacrylamide gel electrophoresis system with one buffer The electrical current is conducted through the buffer and gel by moving ions In the electrodes and external wire circuits current is conducted by electrons Hydrolysis of water at the electrode surfaces will accomplish the conversions between electrons and ions The reaction at the cathode is 2e 2H20gt2OH39 H2 1 with electrons e being supplied by the external circuit and molecular hydrogen being formed and released at the electrode surface The buffer is a mixture of a weak acid and its conjugate base so that the hydroxyl ions will shift the equilibrium to the right resulting in formation of more A HA OHquot lt gt A H20 2 The corresponding anodic reactions are H20 gt 2Hquot39 12 02 2e 3 H3939Aquot ltgt HA 4 Electrons retum to the power supply from the anode and molecular oxygen is released at the anode Any cations in the system will of course tend to be transported toward the cathode thus contributing to the total current ow The use of buffer instead of a simple salt solution in the reservoirs is needed to prevent pH shifts As shown in Figure 1 the protein solution is applied into a well formed at the top of the gel under the upper reservoir buffer Glycerol sucrose or a similar nonionic substance is added to increase the density of the protein solution so that it will form a layer under the upper reservoir buffer When the power supply is activated a voltage gradient is established in the gel and anionic protein molecules will migrate into the gel High pH values are usually chosen so that the majority of proteins migrate toward the anode Cationic proteins will migrate into the upper reservoir and be lost Protein molecules entering the gel will migrate more slowly than molecules still in free solution above the gel because of a sieving effect The protein zone will therefore become slightly narrower and permit greater resolution of the components Resolution of proteins in gel electrophoresis is a function of the thickness of the starting zone i e the volume of the sample For these reasons Ornstein and Davis developed a multiple buffer system for concentrating the proteins in the sample i e reducing the thickness of the starting zone much further still than that achieved by the transition from free solution to gel It is the system of Omstein and Davis that we will use in this experiment This more complicated multiple buffer system is referred to as discontinuous or simply disc electrophoresis The principle is that a discontinuity of pH is established between the leading buffer anions that have high mobility and the trailing anions that have a low mobility In an electric eld this discontinuity results in the concentrating and stacking of proteins When stacked the proteins are in very sharp bands lined up one behind the other according to their electrophoretic mobilities The discontinuity junction can be observed with the eye as a result of refractive index changes caused by sharp concentration changes at the discontinuity junction In the buffer systems used in this experiment a difference in pH is used to change the mobility of a weak acid glycine H3N3939CH2COO The mobility of an ion is the rate at which it moves in an electrical eld The units of electrophoretic mobility are velocity divided by electric eld strength ie cmsecvoltscm or cmz volt 1 sec391 The anionic form of glycine HZNCH2COO has an experimentally determined mobility of 15 x 10395 cm2 volt391 sec391 or 15 mobility units 15 mu where 1 mu 10395 cm2 Vquot1 sec391 When protonated it forms a zwitterion H3Nquot39CH2COO that has a mobility of zero When the pH is at the pKa of the amino group glycine molecules exist half of the time in the anion form and half of the time in the zwitterion form The resulting mobility is the average of the mobilities for the two forms O 152 75 mu The system utilizes two gels and three buffers in the same apparatus described in Figure 1 To separate anionic proteins the running gel also known as the small pore gel is buffered by an amine at high pH often Tris at about pH 9 The large pore or stacking gel which is poured onto the running gel after the latter has polymerized has a lower concentration of acrylamide than the running gel The stacking gel is also buffered with the amine but the stacking gel buffer is selected to have a pH about two to three pH units lower than the pH of the running gel buffer The current carrying anion in the TrisHCl buffers is C1quot The stacking gel buffer is also used in the sample protein solution The upper reservoir solution the third of the buffers mentioned above is again prepared using an amine Tris but the current canying anion in this case is not Cl Instead it is a salt of a weak acid which will have a different charge and hence a different mobility in the stacking gel and the running gel buffer Glycine is the weak acid used in the system under consideration here When the current begins owing in the apparatus described above Clquot ions 37 mu glycinate ions anionic proteins and an added anionic indicator dye bromphenol blue all begin a downward anodic migration in the electric eld Glycinate ions entering the protein sample and stacking gel region however nd themselves in a low pH and become protonated to the zwitterionic form The average mobility of glycine becomes less than that of anionic proteins In this form glycine is one of the slowest anions present For this reason glycine is called the trailing anion V1thin the stacking gel and sample solution the order of increasing negative mobility is trailing ion glycineltproteinsltbromphenol blueltCl39 If the electric eld voltage gradient is constant mobile anions will move out of the stacking gel more rapidly than the less mobile trailing anions will tend to enter However since the current in all regions of the gel must be the same i e all regions are in the same electrical circuit the same number of ions must be transported through every crosssection of the gel In any region where the mobility or the concentration of the ions is low the resistance is high so a larger voltage gradient develops The larger gradient then transports these ions more rapidly through the region just compensating for their lower mobility thus the current in all parts of the circuit remains constant The region of high voltage gradient begins at the interface between the upper reservoir buffer and the sample and spreads downward as the chloride ions move out In the regions where chloride ions remain the voltage gradient is small Thus the protein and bromphenol blue 8 molecules tend to migrate more rapidly in the regions of high voltage gradient than elsewhere in the gel The bromphenol blue and protein become trapped in a narrow migrating band between the leading chloride ion and the trailing ion This is the stacking condition It is inherently stable because any bromphenol blue or protein molecule moving more slowly than the stacked zone will enter a region of higher voltage gradient and be retumed to the zone Any bromphenol blue or protein molecule moving more rapidly than the stacked zone will enter a region of lower voltage gradient and be overtaken by the migrating stacked zone The stacking process is represented in Figure 2 parts a and b p y p it an it a A rz ge onccaoaocc at Figure 2 Stacking in discontinuous gel electrophoresis httpnationaldiagnosticscom images1 4 2bgif Under the conditions described in the previous paragraph proteins with different charge in a mixture would separate but because they are so tightly packed under the stacking conditions the separation is of no practical use Even bromphenol blue could not be distinguished from the protein directly behind it However when the trailing ion reaches the running gel where the pH is higher mobility is altered As the ratio of anion to zwitterion increases the average mobility of the trailing ion increases until it becomes more mobile than the proteins present Thus the trailing ion disrupts the stacking process of the proteins and the latter now migrate with mobilities characteristic of their size and charge In order of increasing mobility the anions in the running gel are now proteinslttrailing anionltbromphenol blueltCl SDS Polyacrylamide Gels A number of detergents have been employed to solubilize biological materials One of the most effective of these in solubilizing protein is the anionic detergent sodium dodecylsulfate SDS SDS binds to proteins via both polar and hydrophobic interactions and introduces a net negative charge that helps solubilize proteins in water When detergent is added in excess large numbers of detergent molecules are bound to each protein molecule with a near constant ratio of SDS to protein for most but not all proteins 14 g SDS10 g protein As a consequence of SDS 9 binding a large net negative charge is observed for the detergentprotein complex In fact the charge due to SDS is generally so large relative to that of the ionizable groups on the protein that the charge on the protein due to the amino acid side chain ionization becomes insigni cant and SDSprotein complexes tend to have a similar constant chargesize ratio For this reason SDS gels separate proteins based almost entirely on their molecular size In SDS polyacrylamide gel electrophoresis or SDS PAGE the electrophoretic migration of a larger protein molecule is generally retarded more than the migration of a smaller one due to sieving effects hence the Rm of a small SDSprotein complex will be greater than that of a large one When Rm values are plotted versus the logarithm of the protein molecular size a straight line is generally obtained see Figure 3 Several proteins of known molecular sizes are used to construct such a standard curve Figure 3 With such a curve the subunit molecular weight of an unknown protein can readily be determined SDSPAGE Standard Curve 5A 52 50 quot 8 k6 4 2 400 I I I I I I I 00 Q5 13 15 29 25 3 35 43 MdWWam Figure 3 Standard curve for determining subunit molecular weight of proteins treated with SDS Note large differences in molecular masses of the high molecular weight proteins squares result in small changes in mobility Since SDS also affects noncovalent associations within a native protein tetrameric proteins such as lactate dehydrogenase are converted from an enzymatically active tetramer 140000 MW to inactive monomers 35000 MW Hence gel electrophoresis in SDS measures monomer or subunit molecular weights Le Mr The combination of nondenaturing and SDS PAGE can yield information on both the native molecular weight as well as the subunit composition of a particular protein molecule It should be noted here that there are some limitations to keep in mind when interpreting SDS polyacrylamide gels For example proteins that are conjugated to other substances eg the carbohydrate of glycoproteins give aberrant molecular weight estimates by SDS PAGE since the ratio of SDS to protein may not be 14 1 For similar reasons some very hydrophobic integral membrane proteins may also migrate anomalously in SDS gels Finally proteins having a large net positive charge may also fail to t on the standard curve 10 MCB 140L PROTOCOL 6 PART 1 SDSPAGE GELS AND TRANSFERING Due to time constraints you will not be asked to pour your own SDSPA GE gels For background information I ve summarized the technique Assembling the glass plate sandwich I Assemble the glass sandwich on a clean surface Lay the longer rectangular glass plate down rst Place two spacers of equal thickness along the short edges of this plate Next place the shorter glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are aligned At this point the spacers should be sticking up above the longer glass plate approximately 5 mm 2 Loosen the four screws on the clamp assembly and stand it up so that the screws are facing away from you Firmly grasp the glass plate sandwich and gently slide it into the clamp assembly with the longer glass plate against the front face of the acrylic pressure plate Tighten the top screws of the clamp assembly 3 Place the clamp assembly into the alignment position on the casting stand so that the clamp screws face toward the casting positions Loosen the top two screws to allow the plates and spacers to sit ush against the base Insert an alignment card between the glass plates to keep the spacers in the proper upright position Hold the clamps at the base of the stand and re tighten the top two clamp screws Note failure to use the alignment position to properly orient the glass plates can result in casting leaks while pouring the gel or buffer leaks during the run 4 Remove the clamp assembly from the alignment position and tighten the bottom two clamp screws Check for proper alignment by inverting the gel sandwich and looking at the surface of the two glass plates and spacers All three surfaces should be ush 5 Transfer the clamp assembly to one of the casting positions on the casting stand If two gels are to be cast place the clamp assembly on the side opposite the alignment slot to make aligning the next sandwich easier To attach the clamp assembly butt the acrylic pressure plate against the wall of the casting position at the bottom so that the glass plates rest on the rubber gasket 6 Snap the acrylic plate underneath the overhang of the casting position by pushing with the white portions of the clamps Do not push against the glass plates or spacers as this could break the plates The gel sandwiches are now ready for casting 11 1 START HERE Sample Application to the gel 1 Assemble the prepoured SDSgel into the U shaped electrode assembly that makes the upper inside between the two gels and lower reservoir outside the glass plates compartments Try to seat the gel plate ush against the gasket to ensure there are no leaks Once seated nd another lab group that is also ready to put their gel into the U shaped apparatus two gels are required to build the upper and lower reservoir compartments or you can use a dummy plate 3 Once the gels are seated plate the electrode assembly into the clamping unit and close the cams Place in tank and ll the upper reservoir with reservoir buffer until the level of buffer is above the upper gel Check to be sure that there is no leakage of buffer from the upper reservoir to the lower 12 I 5 quotiFIquot39iJI r391quot39j39iquot5mylt I P 3391L qlty I A V 3quot39I iquot quot iIr quot i5amp7 L 1 39 39 v Remove the comb from your gel very carefully Do this by exing the comb back and forth as you slowly and gently pull the comb free If there is no leakage from the upper reservoir to the lower reservoir then it is safe to ll the lower reservoir with reservoir buffer I Rinse the wells out to remove unpolymerized acrylamide Load your samples into the gel sample wells according to Table 2 13 Table 2 Lane assignments for the SDS polyacrylamide gel One gel per pair Gel Lane Samples Prepared in MURB 1 25 ul TOTAL 80 ug 2 25 ul PMS 80 ug 3 25 ul MITO 80 ug 4 25 ul HSS 80 ug 5 25 ul HSP 80 ug 6 10 pl Prestained protein standards see Table 3 7 25 ul tube 1 50 ug 8 25 ul tube 2 50 ug 9 25 ul tube 3 50 ug 10 Table 3 BioRad Precision Protein Standard BioRad 1610375 or Precision Plus Protein Dual Color Standards 1610374 same mw but different colors Xzsoxo 150 100 Electrophoresis 1 Connect the electrodes to the apparatus Run the gel at 200 Volts 2 Stop the electrophoresis when the dye front is about 05 cm from the bottom Disassemble and rinse the apparatus 3 Pour out the buffer and disassemble the plates according to the instructions for SDS PAGE Transferring of Gel to Membrane Proteins transferred to the membrane are tightly bound to the surface of the membrane through hydrophobic interactions The direction of migration of the proteins to the membrane is perpendicular to the original polyacrylamide gel which results in a reproduction of the pattem of protein bands present on the gel see diagram below Spaces on the membrane not occupied by protein bands will attract and bind any additional protein it comes in contact with For this reason solutions used for subsequent incubations and washes contain salt and a detergent such as I4 Tween20 to prevent proteins used as detecting molecules from becoming nonspeci cally bound to the membrane or to transferred proteins for which they have little af nity To detect a speci c protein on a Western blot monoclonal antibodies are most commonly used Figure 4 Figure 4 Xray lm exposed by light Sandwich antibody detection of an antigen Ag on a Western blot Electroblotting to a Nitrocellulose Membrane for Western Blot Note Wear gloves to avoid nitrocellulose membrane contamination 1 Remove the SDSPAGE gel from the gel apparatus after electrophoresis and place the gel in a small sandwich dish Be sure to cut the stacking gel from your gel using a razor blade Check with the TA if you aren t sure how to do this Add enough transblot buffer 25 mM Tris 192 mM glycine 20 methanol to cover the gel and allow it to equilibrate for 10 min Obtain a precut piece of nitrocellulose membrane Mark your initials on the membrane with pencil but be careful not to touch the membrane with your ngers Wet the nitrocellulose membrane in a small dish with 5 ml water Allow the membrane to hydrate for at least 5 min Remove the water and cover the membrane with transblot buffer Equilibrate for at least 5 mm Fill a dish halffull with transblot buffer and assemble the Bio Rad transblot components in the following order ensuring that the components are fully wetted following each step Note orientation is important Open the gel holder cassette like a book and place it in the dish with the black side down 7 10 11 BioRad transblot gel holder cassette Assemble the sandwich in the following order a First ber pad directly on top the black side of the cassette b Whatman paper c SDS gel d wetted nitrocellulose membrane smooth out all air bubbles e Second piece of Whatman lter paper f second ber pad atop the second Whatman lter paper Holding all components rmly in place close the cassette like a book and clamp it closed Closed gel cassette for transblotting Place the transblot electrode insert into the buffer chamber along with the ice unit and ll the chamber with transblot buffer The assembled cassette can be placed into one of the two slots in the transblot electrode insert Orientation is important The black side of the cassette should face the black side cathode of the insert Put the lid on the chamber such that the proper electrodes are connected to the proper outlets of the power source Use a frozen popsicle for proper cooling Tum the power on for the electrophoretic transfer Set the power at 100 volts constant voltage and run for exactly 30 min Tum off the power and remove the membrane to a plastic dish containing deionized water With assistance from the TA incubate your membrane with Ponceau dye for a few minutes and then rinse in water Note andor use your cell phones to photograph the membrane the differences in intensity of the stained proteins between lanes Be prepared to explain these differences in your notebook 16 MCB l40L LABORATORY 1B PROTEINPROTEIN INTERACTIONS CONTINUATION OF YEAST TWO HYBRID ASSAY We will utilize the twohybrid assay to determine whether the proteins listed in the table from Lab 1 interact in a pairwise fashion The strain of yeast you are using was designed to have three reporter genes integrated into yeast chromosomes Ade2 His3 and LacZ We will use Ade2 and His3 to test interactions between bait and prey fusions see transformation lab 1 To test for interaction we must patch out yeast cells on media which will allow us to determine whether reporter genes have been activated Please follow the directions given below Determining Interactions 1 Obtain the following plates leuuraade leuurahis leuura plates 2 Use a permanent marker to divide the plates into multiple boxes 3 Using a sterile toothpick select 1 colony and streak onto each plate within the square of the patching guide Keep track of the location of each colony in your notebook 4 Give plate to TA s who will place them at 30 for two days 5 After two days you will be responsible for assessing the growth of each transformant on the different types of media see protocol 1C below 1 MCB 140L LABORATORY 1B PROTEINPROTEIN INTERACTIONS CONTINUATION OF YEAST TWO HYBRID ASSAY We will utilize the twohybrid assay to determine whether the proteins listed in the table from Lab 1 interact in a pairwise fashion The strain of yeast you are using was designed to have three reporter genes integrated into yeast chromosomes Ade2 His3 and LacZ We will use Ade2 and His3 to test interactions between bait and prey fusions see transformation lab 1 To test for interaction we must patch out yeast cells on media which will allow us to determine whether reporter genes have been activated Please follow the directions given below Determining Interactions 1 Obtain the following plates leuuraade leuurahis leuura plates Use a permanent marker to divide the plates into multiple boxes Using a sterile toothpick select 1 colony and streak onto each plate within the square of the patching guide Keep track of the location of each colony in your notebook Give plate to TA s who will place them at 30 for two days After two days you will be responsible for assessing the growth of each transformant on the different types of media see protocol 1C below 18 MCB l40L LABORATORY 1B PROTEINPROTEIN INTERACTIONS CONTINUATION OF YEAST TWO HYBRID ASSAY We will utilize the twohybrid assay to determine whether the proteins listed in the table from Lab 1 interact in a pairwise fashion The strain of yeast you are using was designed to have three reporter genes integrated into yeast chromosomes Ade2 His3 and LacZ We will use Ade2 and His3 to test interactions between bait and prey fusions see transformation lab 1 To test for interaction we must patch out yeast cells on media which will allow us to detennine whether reporter genes have been activated Please follow the directions given below Determining Interactions 1 Obtain the following plates leuuraade leuurahis leuura plates Use a permanent marker to divide the plates into multiple boxes Using a sterile toothpick select 1 colony and streak onto each plate within the square of the patching guide Keep track of the location of each colony in your notebook Give plate to TA s who will place them at 30 for two days After two days you will be responsible for assessing the growth of each transformant on the different types of media see protocol lC below 19 MCB 140L LABORATORY 7 PROTEIN LOCALIZATIONWESTERN BLOT Today you will nish analyzing your differential centrifugation and protease protection fractions by Westem blot analysis Westem blotting involves the electrophoretic transfer of protein molecules from a polyacrylamide gel to a solid support such as a polyvinylidene di uoride PVDF nitrocellulose the one we will use or nylon membrane followed by visualization with a either a uorophorelinked or enzymelinked antibody in some cases an enzymelinked proteinligand with a high af nity to a particular protein on the membrane blot can be used instead of an antibody Last lab period we separated proteins in our fractions using SDSPAGE and subsequently transferred them to a solid membrane for Westem analysis For this transfer an electric current is applied to the gel so that the separated proteins transfer through the gel and onto the membrane in the same pattern as they separate on the SDSPAGE We will rst block all sites on the membrane using excess protein in our case milk proteins that do not contain blotted protein from the gel so that antibody serum will not nonspeci cally bind to them causing a false positive result Often the membrane is cut to facilitate testing of different samples with different antibodies To detect the antigen blotted on the membrane a primary antibody is added at an appropriate dilution and incubated with the membrane This antibody will bind to the protein containing the antigen and unbound antibody or antibody nonspeci cally bound to the membrane will be washed away at the end of the incubation In order to detect the antibodies that have bound anti immunoglobulin antibodies coupled to a reporter group such as a uorophore or the enzyme horseradish peroxidase HRP are added eg Goat anti human IgG HRP this is often called a secondary antibody or conjugate In our case we will use a uorescent secondary antibody Finally after excess unbound secondary antibody is washed free of the blot the uorescent antibody can be detected using a highly sensitive CCD camera detection system h p WWWlicorcombioproductsimagjng systemsodysseyodyssey imagenjsp In the case of enzyme linked secondary antibodies such as HRP substrates are added to the membrane that result in a colored precipitate or the production of light To understand more about the latter detection methods please read the appendix to familiarize your self with Enhanced Chemiluminescent ECL Before coming to lab please be sure to calculate the volume of antibodies you will need based on the dilutions listed below pay attention of updates as the batch of antibody used on a given year may change the dilution 20 U3 MCB 140L PROTOCOL 7 WESTERN BLOTTING Incubate membrane for at least lhour in 1X PBS 5 nonfat dry milk 01 Tween Blotto TA 395 will have completed step I for you Find your membrane and transfer them to Western container Add 15 mls of Blotto to each membrane Add X ul of antiPAP lI5000 dilution for salt extraction or antiCdcll dil for the protease 3 protection and X ul 12000 of anti porin sera Xul of antiplasma membrane 1 and Xul 15000 of antiSgtl antibody to membrane and shake for 45 minutes Wash membranes by pouring off Blotto add 15 ml of fresh Blotto to each membrane and shake for 10 minutes Repeat Step 4 Pour off Blotto Add 15 ml of Blotto to each membrane Add 15 ul of antimouse sera to membrane with lanes 16 Shake for 45 minutes 10 Wash membrane repeat steps 4 and 5 11 Rinse Membrane 2 times in dH20 and leave in dH2O 12 The blot will be analyzed on the LiCor detection machine by the TA 21 MCB 140L LABORATORY 1C PROTEIN PROTEIN INTERACTION CONTINUATION OF THE YEAST TWOHYBRID ASSAY During one of the incubations for your Westem Blot obtain your patched yeast two hybrid plates Use the space provided below to create a table describing the growth of the strains on the different media and interpret your data TYPES OF MEDIA INTERACTION Interaction Tested Given the domain structure of Mdvl see diagram below and lecture notes what can you conclude about its interaction with Fisl and Dnml Puiybesic edn39su iii region Ph dnnmn 1 415 Schematic of Cdcll the septin protein used in the twohybrid experiment Some type of table or schematic of the plasmids 22 biochemically distinct elution approaches results in a highly speci c puri cation i e with few contaminants MCB 140L PROTOCOL 8 CDC llTAP PURIFICATION In pairs you will do two TAP puri cations one with IgG sepharose beads and one with sepharose beads immunoprecipitations one with antibody and as a control one without antibody 1 10 ll 12 13 Obtain 50ul of IgG sepharose beads and 50ul of sepharose beads 11 slurry Centrifuge each tube at 1000 x g for 2 minutes This will pellet the beads leaving a translucent pellet and a clear supematant Remove the supernatant with a P200 making sure to not take any beads Moving quickly do not allow the beads to dry resuspend beads in 250 ul of IPP150 buffer see below Repeat steps 1 and 2 2 more times After last wash resuspend beads in S00ul of IP buffer Keep on ice while preparing lysates While beads are washing obtain one aliquot of WCE or HSS fraction from yeast expressing CdcllTAP Add NP40 10 stock and NaCl SM NaCl to the same concentration as the IPP150 buffer SAVE AN ALIQUOT for loading on your gel 80ug Ensure that the extract is at 20mgml nal we will tell you the starting concentration Use IPP150 buffer if necessary to achieve the necessary volume and concentration Supplement extractbead mix with PMSF and benzamidine protease inhibitors by diluting stocks 1 1000 Place diluted lysates on ice for 20 minutes and then centrifuge for 20 minutes on high at 40C This will yield the nal lysates While centrifuging the lysate remove the IPP150 buffer from the beads prepared in steps 14 You may need to recentrifuge 1000 x g for two minutes at room temperature if you disturbed the beads After centrifuging your lysate carefully remove the supernatant being sure not to disturb the pellet what s in this pellet and then add S00ul to each tube of beads Place tubes on nutator in the cold room for 1 hr Pellet your beads at 40C 1000 x g for 2 minutes Remove and SAVE the supematant This is your Depleted Sup It contains all the proteins that did not bind to the sepharose beads Make sure you mark which tubes saw the IgG sepharose Wash beads by resuspending them in 1000ul of IPP150 buffer centrifuge 1000 x g for 2 minutes at 40C 14 Repeat wash step 13 and remove supernatant 15 Wash beads by resuspending them in 1000ul of TEV CB and centrifuge as in step 13 16 Repeat wash in TEV CB 17 18 Resuspend beads in 50 ul of TEV CB and add lul of TEV protease gt20 Uul Incubate 11Sh at 160C with occasional mixing 19 Transfer supernatant to new tube labeled TEV C IgG This represents the cleaved proteins from the IgG sepharose beads 24 20 Wash beads in 50ul TEV CB centrifuge and add to tube with cleavage product Save the beads 21 Add 100 pl of 30 TCA to TEV CB tubes 22 Mix well by vortexing the tubes 23 Place at 60 C for 5 minutes in a temp block located in lab 24 Place on ice for 10 minutes 25 Centrifuge tubes at 4 C for 10 minutes on high 26 Remove supematant and resuspend pellet in 100 ul of acetone The pellet will not completely go into solution but you need to make sure it gets broken up 27 Centrifuge at 4 C for 5 minutes on high 28 Remove supematant and allow pellets to airdry by leaving tubes open on ice for 10 minutes 29 Resuspend pellets in tubes the two TEVC tubes in 25 ul of MURB Place the 80ug of starting extract you saved 80ug of depleted supernatant and the beads all in 25ul MURB and heat 600C for 5 After heating carefully remove the supematantMURB from the beads and put it in a new tube labeled beads 30 You should now have 5 labeled tubes total depleted SIN TEVC TEV C beads Give these welllabelled tubes to the TA for safekeeping Make sure the tubes have the fraction names as well as you group initials for later identi cation Buffers IPP150 10mM Tris HCl pH 80 300mM NaCl 01 NP 40 TEV CB cleavage buffer 10mM TrisHCL pH 80 300mM NaCl 01 NP40 05mM EDTA 10mM DTT or 10uM bME 25 IPP150 Calmodulin Binding Buffer CBB l0mM TrisHCL pH 80 300mM NaCl 1mM Mg Acetate 1mM imidazole 2mM CaCl2 10mM bME IPP150 Calmodulin Elution Buffer CEB 10mM TrisHCL pH 80 300mM NaCl 002 NP 40 1mM Mg3 Acetate 1mM imidazole 20mM EDTA 10mM bME 26 MCB 140L LABORATORY 9 CDC11TAP PURIFCATION SDSPAGE ANALYSIS Today you will analyze the fractions from the TAP puri cation on SDSPAGE and transfer the proteins separated by SDSPAGE onto membranes for Western Blotting analysis in the following laboratory Refer to Laboratory 6 for protocol details Load your samples into the gel sample wells according to Table 1 Table 1 Lane assignments for the SDS polyacrylamide gel One gel per 4 students Gel Lane Samples Prepared in MURB 1 25 ul of Total Extract 2 25 ul of depleted SN 3 25 ul of CB Eluate 4 25 ul of CB Eluate 5 10 ul Prestained SDS MW Kaleidoscope Standards see Table 2 cut in middle of markers 6 25 ul of Total Extract 7 25 ul of depleted SN 8 25 ul of CB Eluate 9 25 ul of CB Eluate 10 25 ul of beads from one group Before you store your membrane write your name in the top right hand comer and label the markers Additionally using a scissors cut the blot vertically so each pair can have their half We will choose a combination of antibodies to probe the membranes that will shed light on the puri cation and the copuri cation of associated proteins involved in mitochondrial fusion Please consider which proteins you want to identify What are good positive and negative controls What experimental information would you like to gain from your blot What are your expectations 27 MCB 140L LABORATORY 10 IMMUNOPRECIPITATION WESTERN BLOT ANALYSIS Today you will nish analyzing your coimmunoprecipitations by Western analysis Refer to Laboratory 7 for protocol details Primary antibodies Please write down your choices of antibodies to use in this experiment These may vary from the ones that are actually chosen for you in the lab but it is important to have thought through what data you would like to have at the end of the experiment The nal choices will be discussed in lab Secondary antibodies We typically use 1 10000 fold dilution of the infrared uorescent antibodies that will recognize each species of antibody we use Calculate the actual volumes based on 50mls necessary for the whole class 28 MCB 140L LABORATORY 11 LIGHT MICROSCOPYKOEIILER Introduction The past decade has been witness to an enonnous growth in the use of optical microscopy in cell biology The development of novel uorescent probes such as the Green Fluorescent Protein has attracted diverse investigators to use microscopy in their research These developments coupled with the enormous advances in digital imaging have enabled investigators to describe in a rapid and quantitative marmer the complete behavior of sub micron structures within live cells In this laboratory period we will learn to adjust a microscope for best performance by aligning various optical systems that create contrast in transparent cells In Experiments 1519 we will use these microscopes for uorescence microscopy Before you attempt to use our light microscopes you should familiarize yourself with the names assigned to each part of the microscope and their respective functions Figures 1 and 2 label the major working parts of the Nikon microscopes you will use in this laboratory Specimen glass slid Specimenlolder g 1 Mechanical ggge T i Hexagon Wrench M icoarse Torque die Rs Substage g T T 39 zL Fl rK l C idenser Centering Screw K M i M 39 E If Lama HOl5i 1 Filter Receptacle Field Diaphr gmpgRin H Arm Rest Lamp Housing Cover Detachlng Button Figure 1 29 Binocular Eyepiece Tube Diopter Ring E i Eyepiece tube yep ace Clamp Screw mtemup ary m E distance scale Refocusing Stopper I it Clamp Ring i r Revolving Nosepiece Coarse Focus Knquot Qy Qy Obiestive F39quot F9 v3 I i i alas Condenser Condenser Aperture Diaphifglri Ring W J c Q j Condenser Carder Ri hi E E condenser Clamp Screw pj p 3 i Brightness Adjuster Power Switch Condenser Eopcu QKnob 0 h Xaxis Stage Motion Control Knob Yaxis Stage lvlotion Control Knob Power Cord The optical path The optical components contained within modern microscopes are mounted on a stable base that allows rapid exchange precision centering and careful alignment between those assemblies that are optically interdependent Together the optical and mechanical components of the microscope including the mounted specimen on a glass microscope slide with a coverslip form an optical path with a central axis that traverses the microscope stand and body The microscope s optical train consists of an illuminator including the light source and collector lens a substage condenser specimen objective eyepiece and detector which is either some form of camera or the observer39s eye Researchlevel microscopes also contain one of several contrast enhancing devices that are often positioned between the condesnor and the sample and a complementary detector or ltering device that is inserted between the objective and the eyepiece or camera For example conditioning devices and detectors work together to 30 modify image contrast as a function of phase which will be demonstrated in this laboratory exercise The illumination section which houses the lamp is designed to e iciently collect as much light as is possible 39om the lamp and focus the light on the object Also some lamphousings contain a parabolic mirror behind the bulb that recovers light that would not normally be focused by the collector and redirects that light into the collector The parabolic mirror can increase the amount of light recovered from a bulb by as much as 50 Illumination is provided by a tungstenhalogen lamp positioned in the lamphouse which emits light that rst passes through a collector lens and then into an optical pathway in the microscope base Other sources of light found in lamphousings include quartz halogen bulbs mercury or xenon arc bulbs and laser illumination that can be used for uorescence microscopy A second set of lenses found immediately below the specimen is called the condenser that forms a cone of illumination that bathes the specimen located on the microscope stage and subsequently enters the objective Condensers on modern microscopes generally contain large discs which allow prisms and apertures used in various contrasting systems to be rotated into position in the light path depending on which type of contrasting system is to be used we will demonstrate phase contrasting system Condensers also contain an adjustable aperture called the condenser aperture that strongly affects both image contrast and resolution A second adjustable aperture that plays an important part in aligning a microscope is found in the illumination pathway generally in the base of the microscope and is called a eld diaphragm There are often places to put lters in the illumination pathway including heat lters that remove infrared light from the illumination beam and thus protect living specimens from overheating and monochromatic one color lters which help reduce chromatic aberration in the optical system where only black and white video or lm recordings are required The viewing section of the light microscope focuses a magni ed image of the specimen on the microscopist s retina The specimen is most often placed on a glass slide that is held in place on the part of the microscope called the stage The stage has controls that allow the specimen to be moved left and right and forward and back such movements are often called xy translation A magni ed image of the specimen is created by a complex lens called the objective Typical objective magni cations are 10x 16x 25x 40x 60x and 100x Several objectives are mounted to the microscope by a rotating nosepiece that allows the objectives to be changed rapidly The image from the objective is further magni ed and focused on the eye by a lens called an ocular or more commonly an eyepiece Most microscopes used today have two eyepieces and are called binocular microscopes Conjugate planes of light Fundamental to the understanding of image formation in the microscope is the action of individual lens elements that comprise the components in the optical train One interesting effect created by the many lenses in a light microscope is that several locations in the microscope can be in focus simultaneously These planes of common focus are called conjugate focal planes There are two sets of conjugate focal planes the eld planes which are observed in normal viewing mode using the eyepieces and aperature planes which require an eyepiece telescope or Bertrand lens used in place of an ocular for viewing Field Planes Aperture planes Lamp or eld diaphragm Lamp lament Object plane Condenser diaphragm Real intermediate image plane Back focal plane of objective retina Exit pupil of eyepiece 31 As an example of the simultaneous visibility of conjugate focal planes consider that the image of a piece of dirt on a focused specimen could lie in any one of the four eld planes of the microscope oater near the retina dirt on an eyepiece reticule dirt on the specimen itself or dirt on the glass plate covering the eld diaphragm With knowledge of the locations of the conjugate eld planes the location of the dirt can be determined quickly by rotating the eyepiece moving the microscope slide or wiping the cover plate of the eld diaphragm Take the time during this lab period to identify the locations of the eld and aperture planes on your microscope Figure 3 displays two raytracing diagrams showing the location of these planes in a microscope under two focusing conditions In the righthand diagram 21 black arrow has been placed at the plane of the eld diaphragm The diagram shows that the image of the black arrow will appear in the object plane in the plane of the eyepiece and on the retina of the eye The lefthand diagram reveals the conjugate focal planes created when a microscope is correctly aligned for Koehler illumination The image of the lament of the bulb appears in the plane of the condenser diaphragm in the back focal plane of the objective and on the retina of the eye The term confocal microscopy is derived from the words conjugate focal planes Objectives and limits of resolution Objectives are marked on their barrels with information concerning their magni cation lens design glass type numerical aperture type of immersion medium thickness of the coverslip for which they were designed and the tube length of the microscope for which they were designed Here is an example of a label found on the barrel of a Nikon objective Ph 2 Plan 16035 160017 Ph 2 indicates that this lens contains a phase ring for phase contrast microscopy and this phase ring will match the phase aperture number 2 in the condenser you will learn more about phase rings in lab Plan 16035 indicates that the objective has a 16x magni cation and a numerical aperture of 035 The numerical aperture is related to the resolving power of the objective by the following equation d 0617 NA where NA n sin 3 d the smallest distance between two objects for which it can be seen that the two objects are distinguishable The equation is based on the Rayleigh Criterion for resolution it39 the wavelength of the illumination light the half angle of acceptance of the objective n the refractive index of the immersion medium OR when the objective NA and the condenser NA are not equal d 122 7xNAcond NAobj Small values of d represent high resolution Better resolution can be obtained by using shorter wavelength light or by using objectives of high numerical aperture The numerical aperture of objectives is found in a range of 02 to 14 The word plan indicates that the objective has a at eld of focus rather than a curved eld of focus in uncorrected objectives 32 Other words may appear in this area including Planapo Neo uar oelglyand W Planapo means that the objective is corrected to focus three colors of light in the same plane making these objectives particularly suited for color photomicrography Neo uar refers to objectives made of uorite lens elements which will pass light farther into the UV than conventional glass Neo uars are particularly useful in uorescence microscopy because they will pass the excitation light for DNA stains such as DAPI Oel gly and W refer to oil glycerine and water respectively and indicate the type of immersion medium to be applied between the front element of the objective and the coverslip The next line is 160017 which refers to the tube length of the microscope and the thickness of the coverslip in millimeters respectively A coverslip of 017mm is considered standard and is assigned a thickness number of 15 Many microscopes have a tube length of 160mm and as a result objectives made by one manufacturer can be used on another manufacturer s microscope For example an Olympus objective will work well on a Nikon microscope There are also markings on the condenser for each stop on the turret used to position contrasting prisms and apertures For example the phase contrast positions are labeled 2 or 3 while the positions used for Nomarski differential interference contrast DIC microscopy are labeled with Roman numerals I or II Dark eld stops if present are usually labeled D The most variety is found in the labels applied to the bright eld positions on the condensers which are labeled J on Zeiss microscopes H on Leitz microscopes and BF for bright eld on many Japanese microscopes Recording Devices The recording section of a light microscope can create a permanent record of the microscopic image on paper photographic lm or digitally Images can be projected on the hn plane of a 35mm photographic camera or onto a camera with a chargedcoupled device CCD which is a photon detector that is divided up into thousands or millions of picture elements or pixels that store the information from incident photons comprising the microscope image Most research light microscopes are built for photomicrography and include a separate port to accommodate a camera The microscopist can direct light to the eyepieces for direct observation or move a beam splitter into the optical axis of the microscope and direct the light to the camera 33 IMAGEFORMING LIGHT PATH Conjugate field planes Retina Eye Eyepiece Intermediate In 4 Fi 39 39 39 39 image eyepiece j J Objective lens Object plane Stage Condenser lens Field stop diaphragm Collector lens quotW Lamp Lrquot39l ILLUMINATING LIGHT PATH Conjugate aperture planes lris diaphragm of eye 39 4quot 3 Back focal plane of objective 4 Front focal plane of condenser G Lamp filament Figure 3 The locations of the conjugate planes in a light microscope adjusted for Koehler illumination The lefthand diagram shows that the specimen or object plane is conjugate with the real intermediate image plane in the eyepiece the retina of the eye and the eld stop diaphragm The right hand drawing shows the lamp lament is conjugate with the aperture planes at the front focal plane of the condenser the back focal plane of the objective and the pupil of the eve 34 MCB 140L LABORATORY 11 PROTOCOL LIGHT MICROSCOPYKOEHLER Part I Using the microscope STEPI FOCUS ON AN OBJECT 1 You have been provided with samples of diatoms see diagram below Later you will be making preparations of your own buccal epithelial cells 2 Place the slide on the microscope such that the objective lens is centered over the coverslip Start with a low magni cation 10X objective 3 Turn on the illumination and attempt to focus on the slide It often helps to move the microscope stage back and forth in a short oscillating motion as you focus A moving object is much easier to detect than a stationary one 4 Once the objective is focused on the object it is necessary to focus and adjust the condenser lens in the illumination path as described below STEP II Focus the light source on the object 1 After you have focused on an object the illumination source is focused on the object Correct alignment of the illumination path is called Koehler Illumination 2 With the object in focus and the illumination on close the eld diaphragm to its smallest aperture Focus the image of the eld diaphragm by turning the condenser focusing knob 3 Center the image of the eld diaphragm using the two condenser position adjustment screws then open the diaphragm to illuminate the entire eld There are at least two types of scopes in the lab that have slightly different con gurations with regard to adjustment screws Please familiarize yourself with each type of microscope 4 Check the condenser and see that it is on the bright eld setting 5 Replace one of the eyepieces in the microscope with the focusing eyepiece or telescope Close the condenser aperture and focus the telescope until the condenser aperture appears in focus Now open the condenser aperture until it just leaves your eld of view in the back focal plane of the objective The microscope is now correctly aligned Take a moment and experiment with the condenser aperture opening and closing it How do the contrast and resolution of the image you see change as the condenser aperture is changed 19 STEP III Switch to a higher magni cation objective 1 With the microscope correctly aligned and focused on an object simply swing the next higher magni cation objective into place You do not need to adjust focus or anything on the microscope before moving to the next objective because objectives from the same maker eg Zeiss Nikon Olympus etc will be parfocal which means they all focus at the same point even if you switch from a high and dry objective to one which is immersed in oil 2 Observe the specimen and make the necessary small adjustments to focus using the fme focusing knob 35 3 Check that the eld diaphragm is still centered and in focus small differences in lenses may result in incorrect alignment and adjust the condenser focus and aligmnent 4 Check that the condenser aperture is centered and opened to the edges of the back focal plane of the objective The condenser aperture always must be adjusted after changing objectives Part II Resolution tests The resolution limit for an objective and condenser combination can be calculated from their numerical apertures and the wavelength of the illuminating light The resolution limit can also be experimentally determined by using frustules of diatoms as reproducible test objects The frustules have distinct pattems of regularly spaced surface markings striae and pores each with known centertocenter spacings On the prepared slide the frustules of eight different diatoms are arranged in approximate order of decreasing center to center spacing of the markings The names of the diatoms and the number of striae per 10 pm from which the center to center spacing can be calculated are given on an attached sheet from Carolina Biological Work with bright eld optics and start with a lowpower objective and progress upward At each step note the magni cation the NA and the type of objective Remember to refocus the condenser and reset the condenser aperture with each change of objective Reduce the condenser aperture diaphragm opening slightly to increase contrast Obtain the best tradeoff between resolution and contrast to detect the surface markings on the frustules For each objective determine on which diatoms the frustule markings can be detected and hence the smallest centerto center distance resolved by the objective 36 Diatoms in Permanent Dry Mount Species Specimen Amphipleura pellucida Frustulia rhomboides Pleurosigma angulatum Surirella gemma Nitzschia sigma Stauroneis phoenocenteron Navicula lyra Gyrosigma balticum Approximate Length Approximate Width of Frustrule m 80140 50 150 100 200 150 160 280 of Frustrule m 9 10 20 40 3540 1020 3540 3540 3540 Striation Spacing Number of striae per 10 m 37 34 19 20 23 14 8 15 This information is based on information provided by the Carolina Biological Supply Company regarding quotB 25 D Diatom Test Plate 8 formsquot slide mLm 37 Navicula Iyra A j p jj 0 x Srrrmae 1 umf A I P0 777 1 all zoom spac iing 9 quotl39 39 39 V I l l quotI w I l I v 39 n 1 l I r T 1 j m 39 v I 1 H 1 I l l 4 0 1 K 1 39 IL Part III Preparation of buccal epithelial cells Microscopes create images of very small objects by improving on the human eye in three ways First microscopes resolve ne detail well below the limit of resolution of the eye second they magnify the image so that the resolved detail can be seen by the eye and third they create contrast in transparent objects such as cells by amplifying small changes in light as it passes through cells You have examined certain aspects of magni cation and resolution Now you will add lters apertures and prisms to the light path of a microscope which will exploit the subtle optical properties of cells to create greater contrast The types of contrasting systems you will work with are dark eld microscopy and phase contrast microscopy The use of contrasting systems places a premium on cleanliness in the optics of the microscope because the devices which amplify contrast in cells will also amplify the presence of dust particles in the microscope Microscopists often use their own buccal epithelial cells as test samples to align microscopes Buccal epithelial cells are sloughed from the inside of your mouth into saliva and all that you need to do is transfer a small amount of saliva from your mouth to the surface of a slide and then cover the saliva with a coverslip It may not surprise you to hear that such a preparation is called a spit cell preparation The volume of saliva needed to make such a preparation is quite small in the range of Sul to 25 ul You may not actually have to spit on the slide it may be possible to transfer enough spit to the slide by just touching your tongue to the slide Part IV Phase contrast Microscopy Image formation in the light microscope is the result of interference of light scattered or diffracted by the specimen with the undiffracted illuminating light at the image plane The phase ring in the condenser illuminates the specimen with a cone of light The cone of light is captured 38 by the objective and superimposed exactly on a phaseadvancing ring in the back focal plane of the objective The light striking the object is refracted and does not pass through the ring in the objective A typical biological specimen will phase retard light by a quarter of a wavelength The phase ring advances the background illumination by a quarter wavelength The sum of these two effects is that the light passing through the object is retarded by a half wavelength relative to the light passing through the background and the two beams destructively interfere with each other Destructive interference creates a dark object on a bright background Phase contrast microscopy was an important invention because it was the first convenient method for creating contrast in living cells without the use of dyes This invention played such an important role in cell biology that Fritz Zernike received the Nobel Prize for its discovery Phase contrast microscopy remains one of the most popular contrasting systems available because it is easily adjusted and is less sensitive to dirt and imperfections in the optical system than other contrasting systems The great advantage of phase contrast microscopy is that all one needs to do to align a microscope is to exactly superimpose the ring in the condenser with the ring in the objective by the following protocol 1 Select a low magni cation objective l0x or 16x and be sure that it is a phase objective by looking for the phase ring designation Ph 1 for example on the objective Place a buccal cell sample on the stage of your microscope and adjust for Koehler Illumination 2 Replace one of the eyepieces in your microscope with a telescope and focus the telescope until a ring comes into focus This is the phase ring on the back element of the phase objective 3 Turn the turret on the condenser until you come to the position that matches the phase number on your objective 1 for example This turret position contains a phase illumination ring that corresponds to the ring in the objective 4 Using the adjustment lever and disc on the condenser move the condenser ring while observing through the telescope until the two rings are exactly superimposed If the condenser adjustment lever is difficult to move do not force it This lever has a lock and you merely need to tum the grip on the lever to release the lock Look through the normal eyepiece where you should see wellcontrasted cells Play with the alignment of the rings to familiarize yourself with the relationship between object contrast and phase ring alignment 5 You should be impressed with how important and easy it is to align a phase contrast system correctly Other manufactures of microscopes con gure the condensers differently for phase ring alignment You should look at the manufacturer s manual for each brand of microscope to determine ring alignment procedures Part V Dark eld Microscopy The dark eld microscope produces high contrast images of ne structural details within a specimen i e from relatively high spatial frequencies with relatively discrete edges In the dark eld microscope the specimen is illuminated by a hollow cone of light from the condenser as in phase microscopy However the divergence of the illuminating cone of light exceeds the NA of the objective The undiffracted illuminating light is not collected by the objective and relayed to the image plane Thus the background has zero intensity and the contrast is very high You will create the dark eld effect on your microscopes 39 1 Place your buccal cell preparation on the microscope and align the microscope correctly for Koehler Illumination The buccal epithelial cells are transparent and quite difficult to see however you may focus the objective on the edges of air bubbles in your preparation 2 With the 40x objective in place tum the condenser turret to the position marked DF 3 You should see a dark eld image of your saliva with both buccal epithelial cells and bacteria evident 4 Close the eld diaphragm and observe that it is difficult to see the edges of the diaphragm Obtaining Koehler illumination at high magni cation would be quite dif cult using this system but you will discover that systems speci cally designed for dark eld compensate for this problem Examine the diatoms using both phase contrast and dark eld microscopy 40 MCB 140L LABORATORY 12 Direct Fluorescence Microscopy analysis of yeast mitochondrial morphology mutants The Fluorescence Microscope Background In uorescence microscopy the sample you want to study is itself the light source The technique is used to study specimens which can be made to uoresce The uorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a speci c wavelength A uorescent microscope is designed to irradiate the specimen with an excitation wavelength of light that induces a weaker uorescence light to be emitted and then capture the uorescent light to create an image that re ects the levels and position of the uorescent molecule To do this the microscope has an excitation lter that only lets through radiation with the desired wavelength to cause your material to uoresce The resulting excitation of electrons to a higher energy level and their subsequent relaxation emits light The emitted light which is of lower energy and longer wavelength is rst separated from the much brighter excitation light using a barrier lter Fluorescent areas in your specimen can be observed as bright regions against a dark background a very high contrast con guration Principle of Fluorescence 1 Energy is absorbed by the atom that becomes excited 2 The electron jumps to a higher energy level 3 The electron drops back to the ground state emitting a photon or a packet of light the cause of uorescence 100 Excitmriarz l Figure 1 Typical light led Spectra of the uorescent Fhsarvescemre moleculeFITC Intensity C 400 F 500 600 Wavelength nm 41 Basic Optics of a Fluorescence Microscope Re ected light uorescence microscopy is overwhelmingly the choice of today39s uorescence workers This mode of uorescence microscopy is also known as incident light uorescence epi uorescence or episcopic uorescence The illuminator usually a mercury bumer is designed to direct light onto the specimen by rst passing the light through the microscope objective on the way toward the specimen and then using that same objective to capture the light being emitted by the specimen diagrammed in Figure 2 This type of illuminator has several advantages the objective rst serving as a well corrected condenser and then as the image forming light gatherer is always in correct alignment relative to each of these functions most of the unwanted or unused excitation light reaching the specimen travels away from the objective the area being illuminated is restricted to the area being observed and the full numerical aperture of the objective is utilized Reflected Light Microscopy lllumlnator The light travels along the illuminator parallel to the table top and perpendicular to the optical axis of the microscope The light passes through collector lenses and a variable centerable aperture diaphragm and then through a variable centerable eld diaphragm It is incident upon the excitation lter that selects those excitation wavelengths that are wanted to reach the specimen and blocks the wavelengths not wanted to reach the specimen The selected wavelengths reach the dichromatic beamsplitting mirror This mirror is a special type of interference lter that ef ciently re ects shorter wavelength light and ef ciently passes longer wavelength light The dichromatic beamsplitter also sometimes called the dichroic mirror is tilted at a 45 degree angle to the incident excitation illumination and re ects this light at a 90 degree angle directly through the objective and onto the specimen The uorescent light emitted by the specimen is gathered by the objective now serving in its usual image forming function Because the emitted light consists of longer wavelengths it is able to pass through the dichromatic mirror Any scattered excitation light reaching the dichromatic mirror is re ected back toward the light source Before the emitted light can reach the eyepiece it is incident upon and passes through the barrier or suppression lter This lter blocks any residual excitation light and passes the desired longer emission wavelengths toward the eyepieces In most re ected light uorescence illuminators the excitation lter dichromatic mirror and barrier lter are incorporated into a device called a lter cube The more sophisticated systems accommodate 42 three or four uorescence cubes on a revolving turret or in our case on a slider and permit the user to attach replacement custom made exciters barrier lters or dichromatic mirrors Anatomy of a lter cube As stated above in a uorescence microscope a dichroic mirror is used to separate the excitation and emission light paths an essential function given that within the objective the excitation and emission light share the same optics The dichroic mirror39s special re ective properties allow it to separate the two light paths Each dichroic mirror has a set wavelength value cal Fluorescence Emission Fluorescence I Filters Barrier lE l335 39l quot Threaded Fitter Retainlng quot9 g quot39 quot u I E Incoming cams e Mlngr Light waves 0 P A Figurez 0 lcal Block liter Cube Excitation Filter Figure 3 Anatomy of a lter cube the transition wavelength value which is the wavelength of 50 transmission The mirror re ects wavelengths of light below the transition wavelength value and transmits wavelengths above this value This property accounts for the name given to this mirror dichroic two color Ideally the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission The dichroic mirror is a key element of the uorescence microscope but it is not able to perform all of the required optical functions on its own Typically about 90 of the light at wavelengths below the transition wavelength value are reflected and about 90 of the light at wavelengths above this value are transmitted by the dichroic mirror When the excitation light illuminates the sample a small amount of excitation light is re ected off the optical elements within the objective and some excitation light is scattered back into the objective by the sample Some of this quotexcitationquot light is transmitted through the dichroic mirror along with the longer wavelength light emitted by the sample This quotcontaminatingquot light would otherwise reach the detection system if it were not for another wavelength selective element in the uorescence microscope an emission lter Excitation and Emission Filters Two lters are used along with the dichroic mirror Excitation lter In order to select the excitation wavelength an excitation lter is placed in the excitation path just prior to the dichroic mirror Emission lter In order to more speci cally select the emission wavelength of the light emitted from the sample and to remove traces of excitation light an emission lter is placed beneath the dichroic mirror In this position the lter functions to both select the emission wavelength and to eliminate any trace of the wavelengths used for excitation These lters are usually a special type of lter referred to as an interference lter because of the way in which it blocks the out of band transmission Interference lters exhibit an extremely low transmission outside of their characteristic bandpass Thus they are very ef cient in selecting the desired excitation and emission wavelengths 43 Digital Imaging For the past fty years the primary medium for photomicrography has been lm which has served the scienti c community well by faithfully reproducing images from the light microscope It has only been in the past decade that improvements in electronic cameras and computer technology have made digital imaging cheaper and easier to use than conventional photography This section reviews the fundamental concepts involved in digital imaging Digital CCD cameras contain a chargedcoupled device CCD which is a photon detector that is divided up into millions of picture elements or pixels These elements store information from incident photons originating and comprising the microscope image The image can be reconstructed by a computer and almost instantaneously displayed on a monitor for the scientist to examine Because of their incredible dynamic range CCD cameras can produce better resolution of light intensity than lm cameras Their spatial resolution is also comparable The combination of a microscope CCD camera and computer with imaging software has become know as a digital imaging system In 140L we will utilize a digital imaging system to analyze and record data obtained from experiments 1519 We will also learn in detail the anatomy and basis of a CCD camera Fluorescent proteins have revolutionized cell biology The discovery of the Green Fluorescent Protein GFP from the bioluminescent Jelly sh A Wctoria has revolutionized many areas of cell biology and biotechnology because it is able to self catalyze the formation of its own uorophore Thus one can engineer genetically GFP fusions to proteins and targeting signal and express and visualize their localization directly in vivo GFP mutants with blue cyan and yellowish emissions are now available although none with emissions longer than 529nm ie red Recently however a dsRed commercial name protein from a Discosoma specie was identi ed with emission maxima in the red region of the light spectrum The existence of these two proteins enables investigators to determine the localization of two and three components in live cells simultaneously referred to as double and triple label experiments which was not previously possible 44 Yeast as a model organism In the next several labs we will utilize the simple eucaryote S cerevisiae a budding yeast as our model system see httpwwwyeastgenomeorgfVLyeasthtml for more details and information Some of the properties that make yeast particularly suitable for biological studies include rapid growth dispersed cells the ease of plating and mutant isolation a wellde ned genetic system and most important a highly versatile DNA transformation system Unlike many other microorganisms S cerevisiae is viable with numerous markers Being nonpathogenic yeast can be handled with little precautions Large quantities of normal bakers yeast are commercially available and can provide a cheap source for biochemical studies Unlike most other microorganisms strains of S cerevisiae have both a stable haploid and diploid state Thus recessive mutations can be conveniently isolated and analyzed in haploid strains The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques Structural genes corresponding to virtually any genetic trait can be identi ed by complementation from plasmid libraries Plasmids containing genetically engineered modi cations of genes such as a GFP fusion can be easily introduced into yeast cells either as replicating molecules or by integration into the genome Understanding the Cell Cortex and Septins using a Simple Model Organism The fundamental question underlying our studies of septins is the cell cortex is organized into speci c domains Classic examples of distinct cortical domains in cells include the apical and basolateral membrane domains of epithelial cells the prepostsynaptic membranes of neurons the leading edge of migrating cells and the cleavage furrow that forms during cytokinesis in dividing cells Remarkably the site of cortical budding which eventually becomes the cytokinetic plane in budding yeast is an excellent example of a highly specialized cortical domain that informs our understanding of cell cortexes in more complex cells The entree into the regulation of the cell cortex like many other areas of basic cell biology resulted serendipitously from a genetic screen The screen was designed to identify mutants that affect the cell division cycle CDC Researchers screened for mutants that not only failed to grow under restrictive conditions usually high temperature but also for morphological signs that the mutation arrested cells at a particular point during the cell cycle This was possible because budding yeast demonstrate morphological changes in bud shape that more or less correspond to the cell cycle Mutants that enrich for unbudded cells were shown to be arrested in the G1 stage of the cell cycle eg CDC and CDC28 and mutants that arrest with elongated buds are defective for mitotic exit eg CDC3 CDC10 or CDC11 This seminal work and the subsequent characterization of these genes in the regulation of the cell cycle led to a Noble Prize for Hartwell Tim Hunt and Sir Paul Nurse Hartwell and colleagues showed that mutants in CDC3 CDCIO CDCII and CDCI2 gave rise to cells with elongated buds with multiple buds and multiple nuclei see Figure 1 This led to the conclusion that these genes were involved in cytokinesis but little was understood about the molecular role of these genes until their cloning and characterization in the late 1980s Septins Form a Diffusion Barrier Differentiation of new cell types from stem cells requires asymmetric cell fate one cell remains a stem cell and one cell differentiates Fundamental to this idea is the ability of cells to 45 asymmetrically distribute key factors to distinct parts of the cell A key advance in understanding how the cell does this came from studies in budding yeast In a series of elegant experiments Takizawa and colleagues found that an mRNA the translation machinery and the protein product of the mRNA encoded by the IST2 gene all occurred in the daughter bud the smaller one Remarkably the Ist2 protein a transmembrane protein could not diffuse into the mother bud the larger one In septin mutants this diffusion barrier was eliminated and the Istl protein diffused into the mother bud3 However not all cortical proteins are blocked from diffusing actin patches can freely move between mother and daughter bud domains3 It remains mysterious how the cytoplasmic septins form peripheral contacts with the membrane to form a selective diffusion barrier R Figure 1 Mutants with elongated buds and multiple nuclei leading to the conclusion that these are cell division cycle mutants that affect cytokinesis The Molecular Organization of Septins Unlike other cytoskeletal elements it has been challenging to understand the rules that govern septin lament assembly In part this is due to the complex mixture of different septin subunits the peripheral interaction with membranes and the complex behavior of mutants Puri cation of septin proteins demonstrated that the assembly unit probably consists of 46 distinct subunits that can assemble endtoend to form an apolar polymer Cdcl 1Cdcl2Cdc3Cdc10Cdc10 Cdc3 Cdc12Cdcl l 4 5 5 The exact composition of the lament appears to be exible as deletions of subunits still allow for intersubunit interactions lament assembly and viability Septins are dynamic inside the cell This is most dramatically observed during the course of the cell cycle Septins form a ring at the bud neck that is speci cally disassembled in the mother cell after completion of cytokinesis and then reassembled Interestingly the new septin ring is assembled axially to the previous division site in haploids but in a polar position in diploids As the new cell enters the cell cycle the septin ring is positioned at the site of bud growth throughout S phaseG2 In mitosis the septin ring thickens fonning a collar at the division plane In anaphase the collar splits to form two distinct septin rings with a resolvable clear domain between this site is where actin contracts and the cell ultimately divides Figure 2 Using the technique of uorescence recovery after photobleaching F RAP septins proteins exchange at the neck most rapidly during cytokinesis and during the subsequent establishment of the bud site In contrast septins in the ring once the bud initiates growth are stable7 The exchange of septin subunits at discrete times during the cell cycle suggests that there are speci c pathways that control septin assembly and turnover and that stable rings are closely associated with membrane deposition during polarized cell growth Regulation of Septin Dynamics The exchange of septin subunits at speci c times during the cell cycle and the dramatic reorganization of septin rings suggests that there are biochemical switches that control the fate of septin complexes laments As with other cytoskeletal structures biochemical changes that are 46 hourglass intrinsic to septin proteins as well as externally mediated 3 changes have been proposed to control septin dynamics Interestingly septin subunits have amino acid homology with GTPases and recombinant proteins are fully saturated with guanine nucleotides Mutations that are predicted to alter the GTPase domain prevent interaction between septins and other proteins and subtly alter septin ring formation in cells3 Thus it is possible that subunit interactions or trans regulators of the GTPase activity can alter the ability of septins to interact with membranes or each other Importantly the assembly of septins into laments is linked with their interaction with membranes suggesting that regulation of septin GTPase activity directly or their interaction with membranes may be suf cient to drive assembly However effectors of GTPase activity or membrane interaction remain elusive Figure 2 The septin cycle Green represents a fusion between Cdcll and GFP and red represents a fusion between Tubl and CFP a Regulation of septin mction by trans acting Ths hourglass image is fr0m 3 0611 factors is supported by the observation that septins aPPT quotim3e Y in m taPha5e b The themselves are targets of multiple post translational elongation of the anaphase spindle is apparent modi cations septins are reported to be with very little change in the hourglass shape C Spindle breakdown is accompanied by phosphorylated SUMOylated39 and acetylated A kinase Separation of the septin rings that continues d network that likely 1mt1ates Wlth the cyclm dependent until disassembly e and reassembly of a new kinase Cdk Cdczga directly modi es septins but 3130 ring in the mother cell f S Hansen and KB activates the Gin4 kinase which separately modi es Kaplanz Unpublished results septins9 Cdc28 separately targets the Rho GTPas Cdc42 and it39s regulatory GAPS involved in septin ring formation in at the bud site Although phosphorylation sites are not strictly conserved they tend to lie at the interface that mediates heteropolymer assembly suggesting that phosphorylation may regulate assembly Even more poorly understood are the modi cations of septins with SUMO or by acetlyation In the case of SUMOylation septins become modi ed in mitosis although mutations of SUMO sites have only subtle disassembly phenotypes The array of regulatory modi cations and lack of clear connection to lament assembly argues that there are as yet to be discovered signaling connections between septins and other cellular machinery ie chromosomes cytoskeleton polarity signals etc One intriguing possibility is that the role of septins during cytokinesis is tied into the pathways that control chromosome segregation and coordination is necessary to ensure that membrane deposition abscission is coordinated with clearance of chromosomes from the midzone site of cytokinesis Consistent with this possibility is the observation that chromosome passenger proteins associated with the Aurora B kinase are required for the disassembly of the mother septin ring after chromosome segregation Figure 313 14 What other pathways might intersect with septin mediated deposition of cell membranes This will be the topic of your research paper writing assignment Septins Signaling scaffolds vs Membrane regulation and cellular fate 47 How do septins carry out there ftmctions at sites of polarized secretion There are two major functions ascribed to septins i a scaffold that Cdc11GFp mquotta39395r39J 39 39a B allows the interaction of many other cellular A p 3 21 SW5 proteins and ii a regulator of membrane e A A i r dynamics In budding yeast there are over 40 Pc proteins that depend on septins for proper A localization to the motherbud neck Septins are required for the localization of an array of kinases that feedback to govern both cell cycle progression and cytokinesis 5 1517 In addition septins interact with microtubules to ensure proper nuclear position It remains to be understood how the array of kinases and cytoskeletal elements interact to coordinate motherbud neck kymograph a series of 3 timelapse images stacked together Figure 3 A kymograph of septins from metaphase through to the next G1 in wild type ipll321 Aurora B kinase mutant and a depletion of sms the INCENP Ce g139 Wf1 hI m m S 3r ati n and homologue that associates with Aurora B DK Rozelle CYt0k1ne515 A5 mentloneda Septms 3150 ha 3 and KB Kaplan unpublished clear function in regulating the organization of membranes through their interaction with lipids The establishment of a diffusion barrier between dividing cells or cellular compartments has important implications for asymmetric cell fates19 Recent livecell imaging suggests that septin laments formation is coordinated with Cdc42dependent membrane deposition forming a negative feedback loop that allows the integrated shaping of septin domains and membranes In metazoans septins are found in a wide variety of cell types and membrane positions suggesting that there is conserved membrane mction Septins have been found in phagocytic cups in the spenn annulus in dendritic spines and in polarized epithelium surrounding the primary cilium Thus a wide range of specialized cellular functions are likely to require septin function Given these various functions it is perhaps not surprising that defects in septins have been linked to neurological disorders as well as cancer2133 33 However the precise connections to these diseases remain poorly understood Clearly septins represent a nexus where a variety of cellular functions are coordinated and is therefore an area of study that is worthwhile for advancing our understanding of cellular function and the genesis of human disease Thus our journey begins to push back the frontiers of ignorance and make our own contribution to scienti c knowledge REFERENCES 1 Hartwell L H 1971 Genetic control of the cell division cycle in yeast IV Genes controlling bud emergence and cytokinesis Exp Cell Res 692 265276 2 Takizawa P A DeRisi J L Wilhelm J E amp ValeRD 2000 Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier Science 2905490 341344 3 Doyle T amp Botstein D 1996 Movement of yeast cortical actin cytoskeleton visualized in vivo Proceedings of the National Academy of Sciences of the United States of America 939 38863891 48 4 Field C M alAwar 0 Rosenblatt J Wong M LAlberts B amp Mitchison T J 1996 A puri ed Drosophila septin complex forms laments and exhibits GTPase activity The Journal of Cell Biology 1333 605616 5 Frazier J A Wong M L Longtine M S Pringle J R Mann M MitchisonT J amp Field C 1998 Polymerization of puri ed yeast septins evidence that organized lament arrays may not be required for septin function The Journal of Cell Biology 1433 737749 6 McMurray M A BertinA Garcia G Lam L Nogales E amp Thomer J 2011 Septin lament formation is essential in budding yeast Developmental cell 204 540549 doi 101016 j devcel 2011 O2004 7 Dobbelaere J Gentry M S Hallberg R L amp Barral Y 2003 Phosphorylation dependent regulation of septin dynamics during the cell cycle Developmental cell 43 345357 8 CasamayorA amp Snyder M 2003 Molecular dissection of a yeast septin distinct domains are required for septin interaction localization and function Molecular and cellular biology 238 27622777 9 Li CR Yong J Y A Wang Y M amp Wang Y 2012 CDK regulates septin organization through cellcycledependent phosphorylation of the Nimlrelated kinase Gin4 Journal of Cell Science 125Pt 10 25332543 doi101242jcs104497 10 Caviston J P Longtine M Pringle J R amp Bi E 2003 The role of Cdc42p GTPase activating proteins in assembly of the septin ring in yeast Molecular Biology of the Cell 1410 40514066 doi101091mbcE03040247 11 Johnson E S 1999 Cell CycleRegulated Attachment of the UbiquitinRelated Protein SUMO to the Yeast Septins The Journal of Cell Biology 1475 981994 doi 101083 jcb1475 981 12 Agromayor M amp MartinSerrano J 2013 Knowing when to cut and run mechanisms that control cytokinetic abscission TIB Review 128 13 GillisA Thomas S Hansen S amp Kaplan K 2005 A novel role for the CBF3 kinetochorescaffold complex in regulating septin dynamics and cytokinesis The Journal of Cell Biology 1 715 773784 14Thomas S amp Kaplan K B 2007 A Birlp Sli15p kinetochore passenger complex regulates septin organization during anaphase Molecular Biology of the Cell 1810 38203834 doi101091mbcE07030201 15 Barral Y Parra M Bidlingmaier S amp Snyder M 1999 Nimlrelated kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast Genes and Development 132 176187 16 Hanrahan J amp Snyder M 2003 Cytoskeletal activation of a checkpoint kinase Molecular cell 123 663673 17 Longtine M S Theesfeld C L McMillan J N Weaver B Pringle J R amp Lew D J 2000 Septindependent assembly of a cell cycle regulatory module in Saccharomyces cerevisiae Molecular and cellular biology 2011 40494061 18 Kusch J Meyer A Snyder M P amp Barral Y 2002 Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast Genes and Development 16l3 16271639 doi101101gad222602 19 Barral Y Mermall V Mooseker M S amp Snyder M 2000 Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast Molecular cell 55 841851 49 20 Okada S Leda M Hanna J Savage N S Bi E amp GoryachevA B 2013 Daughter cell identity emerges from the interplay of Cdc42 septins and exocytosis Developmental cell 262 148161 doi 101016 j devcel201306015 21 Ihara M Tomimoto H Kitayama H Morioka Y Akiguchi I Shibasaki H et al 2003 Association of the cytoskeletal GTPbinding protein Sept4H5 with cytoplasmic inclusions found in Parkinson39s disease and other synucleinopathies The Journal of biological chemistry 27826 2409524102 doi101074jbcM301352200 22 Takehashi M Alioto T Stedeford T PersadA S Banasik M Masliah E et al 2004 Septin 3 gene polymorphism in Alzheimer39s disease Gene expression 115 6 263270 23 Cytokinesis and cancer 2010 Cytokinesis and cancer 58412 26522661 doi101016 jfebslet201003 044 24 Conquering the complex world of human septins implications for health and disease 2010 Conquering the complex world of human septins implications for health and disease 776 511524 doi101111j1399000420l00l392x 50 MCB 140L PROTOCOL 12 DIRECT FLUORESCENCE MICROSCOPY ANALYSIS OF YEAST SEPTIN MORPHOLOGY In experiments outlined in the next two lab periods we will leam how to operate and utilize uorescence microscopy to answer fundamental cell biological questions We will begin by leaming how to distinguish different mitochondrial morphological phenotypes classify them and quantify an unknown sample that contains an unknown mixture of various classes The following sets of yeast cultures are available to you Please choose one or you may be assigned one for your analysis If you have time you can analyze the other set Set 1 Mid type DMSO 3 hours Wild type nocodazole 3 hours V1ld type onfactor 3 hours Wild type 100mM hydroxyurea 36 hours Set 2 Wild type DMSO 3 hours V1ld type nocodazole 3 hours mad2A DMSO 3 hours mad2A nocodazole 3 hours Each of these strains is expressing a GFP fused to the Cdcll septin protein 1 Aliquot 1 ml of each culture in a labeled eppendorf tube 2 Concentrate the cells by centrifugation in a microfuge at maximum RPM for 2 minutes 3 Resuspend the cell pellet in 100 ul of medium SD ura 3 glycerol 4 Place 2 ul on a microscope slide cover gently with a coverslip and seal with nail polish You can t three samples on one slide 5 Examine the sample and focus on the yeast cells using the IOOX oil objective using phase transmission 6 Illuminate your sample using epi uorescence and the FITC lter cube and observe the shape and distribution of mitochondria in the various cells provided to you NOTE Please examine all of your samples starting with wild type Note your observations and then start to do counts to determine the dominant morphological phenotype count at least 20 cells and express morphologies as de ned in lecture ONLY AFTER THESE STEPS SHOULD YOU START RECORDING IMAGES OF YOUR CELLS You will use these numbers to make a bar graph for your lab notebook and for your writing assignment This should take you the majority of the lab period Once you decide on the representative phenotype then take an image or two Photos are less important to me than proper quanti cation of the phenotypes Compare and discuss your results with your colleagues 51 MCB 14OL LABORATORY 13 Advanced Fluorescence Microscopy The Renaissance in Light Microscopy Lab Objectives 1 2 Appreciate the scienti c direction that led researchers to develop structural methods of lower resolution appropriate for use in living or nearliving tissues Understand the limitations of traditional transmitted light microscopy LM as applied to structures of the size of cells and cellular organelles and understand how uorescence LM confocal and deconvolution microscopy attempt to solve these limitations Appreciate the differences between the confocal and deconvolution methods at both the conceptual and practical instrumentation levels and relate these differences to the relative strengths and weaknesses of the two methods Observe a laser scanning confocal and a wide eld deconvolution in use on the same samples for multicomponent 3D data collection and compare the resulting images Observe 3D computer graphic representations of confocal and deconvolution microscope images and begin to appreciate the issues involved in displaying and analyzing this type of data Develop your intuition about how these methods might be useful for your own research Lab Outline PLEASE NOTE THAT STATIONS MAY CHANGE DEPENDING ON LATEST EQUIPMENT AND VOLUNTEERS DETAILS PROVIDED DURING LECTURE A Station 1 laser scanning confocal microscope 1 3 Note the instrument setup different lasers for different excitation lines laser light brought to scan head where mirrors are used to excite the sample a single point pixel at a time emitted light at all wavelengths collected a single point at a time and assigned the same 3D location as the excitation light all the emitted light passes through an aperature pinhole to reject out of focus components optical elements in the detector separate emitted light according to wavelength separated emitted light regions are collected in different photomultiplier tubes PMTS at the same time Note that the laser scanning confocal image never exists in real physical space but rather is synthesized inside the computer Compare the image visible through the microscope binoculars versus the confocal image presented by the computer Note the differences in image blur between the two and also note the differences in image brightness and dynamic range Observe the effects of varying the pinhole aperature size on the confocal image B Station 2 wide eld deconvolution microscope 1 Note the instrument setup a single mercury arc lamp is used for all excitation wavelengths excitation wavelength for each component is selected using lters in lter wheel excitation light is brought to the microscope via a ber optic cable entire 2D region of interest imaged at same time excitation and emission emitted light passes through a lter in a second emission lter wheel all of the emitted light in focus and out of focus is collected on a CCD camera detector As opposed to the confocal collection of more than one component uorophore requires moving two sets of lter wheels 52 2 Compare the image visible through the microscope binoculars versus the deconvolution image presented by the computer Note how much more image detail seems evident on the computer monitor versus what you noted through the binoculars despite the fact that all the out of focus information remains in the image 3 When you go to Station 3 make sure to see the improvement in image quality following deconvolution C Station 3 computer graphics display and analysis workstation 1 Compare before and after processing images from the deconvolution microscope 2 Compare and contrast 2D optical sections from the confocal and deconvolution microscopes as viewed along the optical sectioning axis and along two orthogonal axes Note the relative strengths and weaknesses of these two methods when viewed in this way 3 View 3D representations of the confocal and deconvolution data Note the assumptions of the methods and the method limitations Answer the following questions as part of your lab write up 1 Why are microscope systems with lower resolution and higher sensitivity light detection an imperative for biologists 2 What are the traditional limitations of light microscopy in terms of resolution and imaging of living cells 3 How do the deconvolution spinning disc and laser confocal systems address these problems a Highlight the differences strengths and weaknesses between the different scopes 4 Why are point spread functions and deconvolution critical for quantitative imaging a How can you determine the number of GFPS in a cell 53 MCB 140L LABORATORY 14 Indirect uorescence analysis of actin morphology using uorescentlylabeled phalloidin mutants Actin laments Factin are dynamic polymers that assemble from monomers subunits in an ATP driven assembly pathway in the cell The assembled Factin can assume a variety of higher order structures that function in the regulation of cell shape changes cell locomotion chemotactic migration and in the polarized transport of secretory and endocytic vesicles and other organelles throughout the cell In yeast actin functions to polarize cells and direct secretory vesicles to the bud site In addition actin has also been shown to play a role in the morphological organization and movement of various organelles such as vacuoles and mitochondria into daughter cells during cell division In yeast Factin laments form structures that fall into two distinct morphological classes patches of cortical actin and actin cables consisting of bundled actin laments Actin cortical patches are invaginations of the plasma membrane around which actin laments and actin binding proteins are organized Yeast has proven to be a valuable system to study the function and organization of the Factin cytoskeleton Indeed there are over 50 proteins in yeast that are dedicated to the regulation of these structures and function in their formation organization and regulation in the cell Fluorescentlylabeled phalloidin has proven to be an excellent tool to study the morphology of the Factin cytoskeleton in a variety of cell types Phalloidin is a toxin from the toadstool quotDeath Capquot Amanita phalloides that binds actin Binding is speci c for Factin Phalloidin is also a very convenient tool to study Factin because uorescent analogs can be synthesized that retain actin binding Phalloidin binds to actin at the junction between subunits and because this is not a site at which many actinbinding proteins bind most of the Factin in cells is available for phalloidin labeling Using different classes of mutants we will evaluate the relationship between septin organization and the actin cytokeleton In the rst class we will use mutations that disrupt septin ogranization to evaluate the effect on actin morphology In the second class we will use mutations that disrupt actin morphology and determine how this affects septin organization To evaluate both structures inside the cell we will use uorescent labels to simultaneously detect septins and actin as described above Set 1 contains mutations known to disrupt septin organization Set 2 contains mutations in regulators of actin polarity For both sets of mutants use the intemet to discuss the types of disruption you expect based on published phenotypic analysis Include a brief summary in your introduction to the lab Set 1 Wild type 370C for 2 hours sgtl I07 370C for 2 hours okpl 5 370C for 2 hours dam1 370C for 2 hours cdc3 6 370C for 2 hours Set 2 V1ldtype LEU2 HIS3 54 tpm1A LE U tpm2A rpm 2 HIS3 slaIA bbc1A LEU HIS3 grows poorly above 25oC Options to avoid xation and spheroplasting Set3 Wild type 2raf gal V1ld type 2dextrose pGALI SL115 2raf gal pGAL1 SLI15 2dextrose pGAL1CTFI3 2raf gal pGAL1 CTF13 2dextrose Set 4 Wild type 370C for 2 hours ndcI0I 370C for 2 hours sgtl 5 370C for 2 hours ipll321 370C for 2 hours MCB 140L protocol 14 Indirect uorescence of actin and septin morphology in yeasts mutants Part I 1 TAs will grow each culture to log phase and then shift temperature as indicated 2 Cells will be xed by adding 1 10 the volume of 37 formaldehyde and xing at the appropriate temperature for 20 minutes 3 Cells are washed with 01M Tris pH 94 10mM DTT for 8 room temp with occasional mixing 4 Pellet and resuspend cells in spheroplast buffer 1 2M sorbitol 20mM KPi pH 74 lter sterilized 5 Pellet cells for 10s in microfuge 6 Resuspend in Zymolyase solution 8mg Zymolyase in 15mls spheroplast buffer 7 Incubate room temperature until 80 of cells have formed spheroplasts 8 Spin spheroplasts gently 4000 rpm for 30 seconds in cold room 9 Resuspend in 12 24 ml ice cold spheroplast buffer 10Each group gets 200ul of spheroplasts 11 Spin spheroplasts gently 4000 rpm for 30 seconds in cold room 12 Resuspend in 50ul of spheroplast buffer with appropriate dilution of antiCdc10 antibody Incubate on rotator for 30 minutes at room temperature 13 Wash cells 3X 05 mls spheroplast buffer spinning as in step 10 each time 14 After last wash resuspend the cell pellet in 50 pl of PBS Add 10 ll of uorescentphalloidin appropriate dilution of antirabbitAlex uor 488 15 Incubate the cells overnight in the dark wrap labeled tubes in tinfoil and place at 4 C until next lab period 55 1Transfer 1 ml of each culture of logarithmicallygrowing yeast cells assigned to you into a labeled microcentrifuge tube Spin down the culture and remove the supernatant 2 Add 0500 ml of 37 Formaldehyde Mix well by inverting tubes 3 Allow xation to occur at room temperature for 30 minutes Mix by taping tubes to the rotating wheel 4 Wash the xed cells by centrifugation in a microcentrifuge on high for 8 seconds Remove the formaldehyde supernatant using a P1000 and discard in supplied waste container Resuspend the cell pellet in 1 ml of PBS Solution 5 Pellet cells by centrifuging as before remove supernatant and add 1 ml of a solution of 01 M Tris pH 9 10 mM DTT to the pellet and resuspend 6 Put tubes on rotating wheel for 10 min at room temperature 7 Wash cells 3X with 1 ml of PBS by centrifugation as in Step 4 8 After last wash resuspend the cell pellet in 50 pl of PBS Add 10 pl of uorescentphalloidin 9 Incubate the cells with phalloidin ovemight in the dark wrap labeled tubes in tinfoil and place at 4 C until next lab period 56 MCB 140L LABORATORY 15 Indirect uorescence analysis of actin morphology in yeast mutants part II Today we will nish the phalloidin and antibody staining of our two sets of mutants BEFORE lab please write down your predictions for what you expect to see in each mutant even if your group did not use that mutant If you feel the result is not knowable then write down TBDto be determined 1 Wash the cells by centrifuging the cells for 8 sec on max speed Remove the supernatant and resuspend the cell pellet in 1 ml of PBS 2 Wash the cells three more times in PBS Solution 3 Resuspend the cell pellet in 2550 ul of PBS Solution 4 Spot 3 ul of washed cell suspension on a slide Place a drop of mounting solution over the cells Cover slide with a coverslip and seal the coverslip to the slide at the edges with nail polish 5 Examine cells using the uorescence microscope Analyze phalloidin and Cdc10 stained yeast cultures using uorescence microscopy for defects in actin or septin organization BEFORE taking images please COUNT the types of septin and actin morphologies you see and compare between control and experimental You will use these numbers to make a bar graph for your lab notebook and for your oral presentations This should take you the majority of the lab period Once you decide on the representative phenotype then take an image or two Photos are less important to me than proper quanti cation of the phenotypes Compare results with your colleagues What conclusions can you draw about the effect of septins on actin and vice versa SAVE YOUR SAMPLES The last lab day will be spent organizing figures and taking extra photos on a first request first serve basis details in lecture 57
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