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Biochemical Methods

by: Dr. Pablo Pollich

Biochemical Methods BIOCHEM 651

Dr. Pablo Pollich
GPA 3.75


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Class Notes
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This 41 page Class Notes was uploaded by Dr. Pablo Pollich on Thursday September 17, 2015. The Class Notes belongs to BIOCHEM 651 at University of Wisconsin - Madison taught by Staff in Fall. Since its upload, it has received 16 views. For similar materials see /class/205196/biochem-651-university-of-wisconsin-madison in Biochemistry at University of Wisconsin - Madison.


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Date Created: 09/17/15
Answers to questions from the March 252009 lecture Insect cells are a popular choice for scientists who want to express large quantities of mammalian proteins If the insect cell system does not produce functional protein then a mammalian cell line can be used but the expression level is usually not as high The cellfree system has a low expression level because the reactions are usually done in about 50100 microliters of cellfree extract so even if the amount ofyour protein produced is high relative to endogenous protein there is just not that much extract Sometimes you can get sufficient protein expression in a system that is not ideal for your protein For example some mammalian proteins express just fine and are soluble and functional in bacterial systems It all depends upon the protein you are trying to express The only way to know ifa protein is going to express in a given system is to try it or read the literature to find someone else who tried it Ifyou are using a recombinant viral system to express protein in eukaryotic cells insect or mammalian cells you first need to get your genome into cells that can produce your virus Getting the genome into cells usually by transfection is very inefficient because the genome is large so most cells will not have a copy of the genome The cells that do get a copy of the viral genome then use it to produce the recombinant virus and that virus can infect other cells in the dish to produce even more virus Depending upon how much protein you need or how many different types of cells you want to infect you may need to make or passage lots ofvirus by taking the initial stock and using it to infect 1020 additional plates of cells Those cells will be infected and then make more virus and so on until you have the amount you need For bacterial expression E coli that has been modified to express T7 RNA polymerase is most commonly used for protein expression For baculovirus expression Sf9 cells derived from the armyworm Spodoptera frugiperda are often used because they are very susceptible to infection by the baculovirus strain used to create recombinant baculovirus Recombinant adenovirus is frequently generated in HEK293 cells derived from Human Embryonic Kidney although once the virus has been made it can be used to infect many different cell types MBL WEEK 3 Head TAs Michael Levine Steve Martin Tumasz Mandy Burnham Student Name DATA SHEET I DATA FOR GROWTH CURVES Time Culture 1 7 Control Culture 2 7 Antibiotic min Dilution I410 CellsmLa Dilution I410 CellsmLa 20 70 90 llO Time Culture 3 7 IPTG Culture 4 7 Lactose min Dilution I410 CellsmLa Dilution I410 CellsmLa 20 50 70 90 llO a Assumptions 1 OD410 015 corresponds to 10 cellsmL 2 Optical density is proportional to cell concentration in log phase Comments from Wed Feb 4 lecture Biochem 65 1 Weibel Q Does the orientation of arrows in molecular orbital diagrams matter A Well sort of Just as long as the two electrons in the same orbital do not have the same orientation recall the Pauli exclusion principle Q Are we expected to remember all of the equations presented in the lecture A Yes Anything I discuss in the lecture you should know Q Can you give an example of a sample exam Is it multiple choice A No I don t have any example I will tell you the format one week before the exam Q I didn t understand the ethylene stuff A Please see the notes I have posted Ifyou are still unclear come see me after lecture Q Are you going to ask us to draw a M0 diagram for a molecule for the exam A Right now you should focus on understanding what we discuss and the lab material and not worry about the exam My policy is that anything I discuss in lecture or that you do for the lab is fair game on the exam Q How does one tell how much heat a certain current will produce A You can predict it from Ioule s law QIquot2 R t where Q is the heat generated I is the current amps R is the electrical resistance of the material ohms and t is time seconds Q What leaves behind the double bond on acrylamide A Well there isn t really a double bond left behind it is just an intermediate when one molecule of bisacrylamid reacts with the growing polymer chain Think about it like this when the polymer chain reacts with bisacrylamide only one of the two CC bonds reacts with the polymer leaving the other CC double bond free This CC double bond can then react with another growing polymer chain which causes two polymers to become cross linked For this reason bisacrylamide is referred to as a 39crosslinker and its function is essentially to connect linear polymers into a connected network Q How do you control the size of the pores in the acrylamide gel Is it the proportions of the acrylamidebisacrylamide ratio A Sort of Typically you increase the amount of acrylamide and increase the crosslinker Q Do we use both agarose and polyacrylamide in slab electrophoresis A Yes they are both commonly used Polyacrylamide is a synthetic polymer which gives you greated control over the physical properties of the resulting gel Q Can you put your lecture notes online TA prelab lectures A Yes they are there I typically upload them on Fri of each week Q Do outside orbitals on the ethylene diagram represent nonhybrid orbitals and inside equal actual orbitals A The outer electrons are the atomic orbital s and the inner are molecular orbitals that depict bonding in this case CC Q Way too much organic chemistry approach eg orbital diagrams electron pushing diagrams Prof Brian Fox doesn t mention anything about orbital or electron pushing A Ifyou don t like this format take it next Fall Q Can someone get electrocuted by an electrophoresis rig in the lab A Not the ones we use You can get a very serious shock but unless you had a precondition you would live As long as you are insulated you are fine that is ifyou stuck your hand in the rig while the current was applied and your feet were immersed in a river then you would be in serious trouble The concern about lethal shocks it to avoid becoming a low resistance path for the movement of electricity Q What are the forces involved in capillary electrophoresis A Here separation is driven by a really fascinating phenomenon referred to as elecroosmotic ow in which the movement of buffer in an electric field actually drags a molecule or protein along with it Very interesting concept Q What wavelength does the Bradford assay use A It is based on the absorbance shift of Coomassie which has a maximum absorption at 595 nm when it is bound to protein A Spectrophotometric Study of Hemoglobin James Ellinger ellingerwiscedu OutlineOverview for today Spectroscopy 0 Hemoglobin Notes for the SPY experiment What is Spectroscopy Study of the interaction between electromagnetic radiation and matter De Broglie Relation A hp Energy ofa Photon E h xv h x The Electromagnetic Spectrum jg i y g 5 398 quotJ 0 quot r a 5 3 6 3 g 3 2 3 5 c 2 1 2 k w gt 3 m 6quot W 10 1 103 Wave length N lW 1 millimeter 1 mete 1 kilometer A 9W 9amp9 Cool Applications Reporter Assays Green Fluorescent Protein Photoelectric Effect Double Beam Spectrophotometer dim mta ng disc gmhngr I minor quot9312 gfz I sm1m Cj sm m thtsoume mnmr gy r md computer minor reference m N Absorbance logIO i I Chan lecorder LambertBeer Law 0 A bec log loll IogT O b is pathlength usually I cm 2 is the molar absorptivity M39lcm39l c is the concentration M O O O T stands for transmittance o If absorbance has multiple components 0 A b Zeiooci Hemoglobin Aw H v H 2 0 2 C H pv H mlH 2 C C C vu CfmC c CCH c e HC CH 1 z a 0 2 H CH wwt cYc cc c C C H CH H K H Home Fepromporphyrin lX Hemoglobin structure 1 o Hemoglobin is a tetrameric protein 0 4 subunits OLZBZ 0 One heme group per subunit 4 moles heme per mole hemoglobin 0 Three redox states are known in vivo met deoxy and oxyhemoglobin Hemoglobin redox states 0 Deoxyhemoglobin amp oxy hemoglobin are the functional forms 0 Methemoglobin is inactive NADH Mullen oglobm 0 Apohemoglobin can also 39 exist in vitro NAD39 o Ascorbate and Sodium 7 K Dithionite will be used to reduce hemoglobin samples Hemoglobin Binding Curve Oxyhaemoglobin Saturation I I I I I 10 20 30 40 50 E0 70 80 90100 PO2 mmHg Pulse Oximeter Pulse Oximeter Used in hospitals 0 Uses ratio of red light to infrared light to measure oxy to total hemoglobin ratio oxygen saturation Measures changing spectra with each pulse as arteries expand 0 Works even if patient is wearing red nail polish Uses 2 wavelengths of light 660 905 nm Pulse Oximeter Used to see if a patient needs supplemental oxygen 0 Limitations 0 Does not measure total oxygen 39 Can t tell if patient is anemic O Reads 85 if there is met heme present 0 Falser high for carbon dioxide or cyanide poisoning 0 There are expensive pulse oximeters that measure carboxy hemoglobin and met hemoglobin that use 7 wavelengths Before coming to lab 0 Read through the experimental protocol Consider making a flow chart for each step in the experiment 0 Review operating procedures for the spectrophotometer 0 Remember the instrument will need to go through it s calibration cycle and warmup Common Errors Lamp not warmed up Wrong Wavelength o Improper blanking Dilution Mistake Dirty or mismatched cuvette Improper seating of cuvette Operation Errors Tips and Considerations 0 Perform experiment in order 2 54 3 o Wipe the cuvettes before inserting them into the spectrophotometer 0 Pay attention to when you use quartz cuvettes and plastic cuvettes Label your tubes when you are performing the dilutions in 5 0 Remember to have aTA sign your DATA SHEET before you leave Calculations Show calculations in lab report 0 One of each kind of calculation 0 Calculated concentrations 0 4 moles heme groups per mole hemoglobin 0 Divide concentration by 4 to get hemoglobin concentration Calculations There should be an Excel worksheet on the course web site for matrix calculations 0 Use cautioust because it doesn t always work 0 Email me if you need help ellingerwiscedu Calculations Report 0 Due Dates 0 Group Feb l3 0 Group 2 Feb 20 0 Reports should include the following 0 Cover Page 0 Optical spectra data sheets and other primary results 39 Clearly labeled graphs 0 Example calculations Watch units and significant figures 0 Answers to questions Clear and Concise smuwwm I as W W 7 9 P SlOW T 19K ms Foo W W 1 Mmsouem n3 and I we t Womanp QJLMPLQ 9 4 I wa 9 W 5LMME Ma aTm FM snnmlqmosl ro m owr if Hull r1 2 Lo WWWa 1 fzfmoTE L rs WIWL 03 DW Hal 924mm MW meswk 3 MM Bum RNAP yaMr wwmm WWW 09m 061 Summeuua gem re r wuvbuces Wuwm 39FW wmw A F MK 10 WW rczmsoa ms 6 39Wmmw aFWWG IS quotth J AWs1um pwmi WW YW tumPS Wm cw Msmmf 51mm 4 W am aw Wm aqmgm W m an M b omzmkw Mucus O39FLauzrwbwi MMWL1LA M 1 Le rujmgu4m 503 n m Ivame W122 Pizeswr L41 may 5 Musmtsw kacl 9 01 wm TDT TE VWLOTEL j w Mom 1 q QUILDSabm 30p 4 H17 0 W a M 8 quotquotquot 9 o139 1b7 15 4W m 10 MW W ww L gm aw Pt rm 3 a sch of 4 6 67W mm DW 01 pkg0 90 m EMo Laws WW 39 WENIi 039 W M ovr YawnD c3 9quot3Mcmzvnst quotISN DS WI PM W lquot W ISSui 0517 M q Wt WWW W 0quot mvhw We 77 16299 mapq 1H1 m mwmr quot77 lS DW NIo McWS DL J M Mg gm39ps m1 m 1 m 945 69 agr MaDswti may m3 Q o l 40k 3391wm L39s MQo lm 739 mutantW wed mn osmi r6m WE lo lt1 mm M W 139 r Smut HWWanL MW LT WIW Mm qkp Raw 3 Kitb 0 HM H7 w Jew w W Hquot 3 L60 0 4 Br q Q 1 WWW H17 3 u Mamasaw I gr A14 113me swam MthS wecssssut TD ukLkWQWWTIK We quota mc W Seawwa W18 m snarkco aF Mt W e Mi alias 39 W WWW WtMT hw j M MWN39Q UFzwrwaTzn g 5 W 5269 wowwand GM 33 wme Btwostms wtv Huh 2 WWI Mm Tm SM MS a gum 1 Wu1zm mmmws 0 we 9W4 A W wLNL 9W Mr 3372 mumm F k meu unwnae 6F M314 wme Aowm w 6F PWD E k PEW Pwm 1M inmqu i wsgw 917 Q GMLAcxorab i L AW 106mm ELME YWULUT anwe A ngm To WWW M HW39V 1 g WNW1U mam PM WWW mausmenod IF A W MM SWPY MS5DA e228 L332 anw as 1mm SKWSS M Ema 2L Wsmm Wk 49w n vs r Latv Wl gkg gt mural unusual cr WYWW To 73m m Quack 11 WA TD WW3 3 J QhA mslbt a qbw quot We ms th 62 c9 WWW 14H WM 2 39 6MWquotquotgt 3 commune n41 owvw Nb aim 39ntL Mews IMPw 719 short WW WWW A Ob A D39Fx ae commw b TO A UW L mmmw ems aw In S Aorl M 39 xg z AESMANQ I aka r MIA WINE Wm MA afffmv xgmntv r 5w m Q39sn 9 119221 I 39 Pwm QM W451 Wt 152 k 01mg IF mewiu Ho ALGA 972 39 9mm um 90 Pct15W s Aemmw 1 Kwi own swagD aquot T39rc w zw TDD EJTELMIAE if 6x2 9 SWCCBYFWWK IL mm m A szD 2amp chmwa 9T1 Vows WNSEL DW W WM TM a Lu ova A 6wquot x m 3 pa w9 ampwg Pm wmeML 6M lTWS EJLw 3mm cam mm Gmtb 217m WsF MMS cad 0 MM LA IL w m aa x56 1 IAN1T9 m ND an 39 CLDIA gtG WNW Nb wax NM 9201 Ewe mm g Cyh mm c cLoAmm D br c r Wt Rotng 5 NW MSW 518m 1 1 W L g 7 3 5w Laws 1c Hilda I i 7 V r H r r 3quot 0W ucmsuuc nwa awaits 7 7 1e72 an 3W teeezylggg V N a N I LvX L 37 Me znb gmu 7 39ng p mm W345i Hm c M WWI1 ooNS IWTWNLS WW wigM2 mam1791395 7 mm 6 Q e L A 4 7 U332 o H 3 isr wwwwhom 69 A4 7 S 3 701 A1 5 112 0 2L lt22 w A gt A 9 613 39 10 D W 194 gt4 W m 0 sir ah Fir o 3 Cd cgt G e 0 i Kr ML 3quothquot 39b45 VS 0 C H39 u mosemn WSL ampISE 6quot139 garW 551 W 122 Qk erMPW do LIWMWIHL no F517 sub 1 gamma Maw Ms L t 3 15 MH m W 43w 199 Swans MW waK amps4 mus O39FF Wyein 01 xi 39 A Wa Wchtgtwgtgt as u robAgg sva MittW ad 124me wabu 1 432211 8141 51W 25 W Wan3 0F szc 09 WM ESr z p Tit1s L 539 Tam ks A Mum Wzd we Argzm a Mange W W 11 5 wqmrm 49 cat8 WMMzk rm Lune Q A J mum U355 4r V W mewm 745a M W M Wags 5W guano A Mme W ms 1sz mmlmm N m wpr W szszg urn 7211 pana A 39 39 Ftva mowPuJms mom 6th 5102 5mm 99 W HWO 13w Mgdtsma powm V w LPl M fv D D 0 A01va 04 If MgCWQw K Max MM mamr Mtqua A a 9013 pm wxs Ham r Msn zsLmaQ YWM b A Tricks for a quot 39 Polvmerase Chain Ni Most primers are about 1522 bases in length It is important to ensure that the melting temperatures Tm of both primers is as close as possible Although there is a formula for calculating melting temperatures I generally assign 4C for Gs and Cs and 2C for As and Ts I prefer to do this especially when primers contain restriction sites The calculated Tm information from companies is misleading as it fails to disregard restriction sites which do not anneal to template When calculating Tm only count the bases that are actually annealing to the template DNA Therefore ignore restriction sites etc Melting temperatures should not be too high or too low If Tm is very low this will result in nonspecific products If Tm is too high it reduces efficiency of amplification Tm between 50 and 60C are ideal E 4 Melting temperatures are affected by the length of the primer and if the sequence is AT or GC rich It is generally advisable to aim for about 50 GC content for each primer and the GC should not differ too greatly between the primers 5 If the two primers have varying Tms I generally choose the primer with the lowest temp and set my annealing temp a few degrees below that For example if the Tm for one of the primers is 58C and the other is 55C I would set annealing temp as 52C This ensures that both primers will be able to anneal to the template 6 Limit 3 GC 7 Avoid hairpins A stable hairpin is formed by at least 3 consecutive bases with a minimum of 3 bases inbetween For example XGGGXXXCCCX This is a stable hairpin and should be avoided However a hairpin such as XGGXXXCCX or XGGGXXCCCX are less stable and can be ignored The more bases inbetween the annealing bases the more stable the hairpin Similarly the more the number of bases forming the hairpin the more stable the hairpin 8 Avoid Primer Dimers Primer dimers are a bigger concern than hairpins and should be absolutely avoided If a hairpin cannot be avoided there is still a chance that the amplification will be successful A stable primer dimer however will not amplify the DNA A stable primer dimer is formed by at least 3 consecutive bases For example this is a stable dimer NNNGACCNNN XXXXXCTGGXXXX The following is not a stable dimer GACTGAGGGCAG AGGAGCCCTTTTC Although there are base pairings along the sequence this is only occurring for 2 consecutive bases at a time and is therefore an unstable dimer D I O O ND I LA 4 When adding random bases before restriction sites avoid potential DAMdcm methylation sequences such as GATC as some enzymes are sensitive to such modi cations Amplifying from plasmid templates is significantly more successful than genomic DNA When setting the Extension time the rule is 1 minute per kb I generally amplify for 35 cycles Too many cycles will increase chances of random point mutations and too few cycles may result in poor amplification Note Shorter DNA fragments will amplify with higher efficiency than long fragments When amplifying very long DNA fragments 612kb it is important to add extra MgC12 in the reaction mixture The best polymerase I have used so far is Long and Accurate Taq polymerase LA Taq 415101 TD 35500 39 mm 0T5 f 4 WW Tuna a Mwm Dbekv as Mad1 3 DamV1 d speak 6 gnawed K39m W109 mwm rte q sot bs MAss mso ssm em rm a qu 39 me m4 L PMM ilk 406 0F bwwd a WW1 754 4 ilk 1011 4 4x agg 9 ILU39 39gfk szg39wmwmms lkm imp I M 1N1 WM akinI1 39 W 9715 mm I mcg 0 Fiver Losu 9 Maine 3 to W zo tava S ua i v awun qs 39 Abucm L1 ggw g NAME ofquot dvbur39n w 9 cr wvm n qu vlw IND WW Fur 39LWMT39 bums quotWm 12an 539 cwqe quot4 ms 4 J euncwsam A was OF 9494 Le W as MMW 91 OFUQH39T esemmm 7mm AM mm 3413 m MIWM N Amman Ms CW axewva so 91141251 Qb M bS WM 6 4 9 quot quotC 7 4 V T neumav 3 Wang we 3 MM m i 9MSz gtf31 WE WT Wiwss sow ii 2 mam 9 W W Via23 o w Draws WmIA P0198 msmswvsd v 11231432 a Dick QJEnTs 82 uum r 11mg N i t 0 WxDMJ39E QueMu mm eta N M Dw omrg HueL11 3mm i Qt SME ToM L SEQ Fm 62an awebana Tr iw us w Wu 2mm m h cFMocmm MM w new 2 9 5 2 x MQSg A w via 2 I 0 L Q A Lk SM mt atbi of W Mow 6m why m FEW F Mk MAL 5 WINTED MN WaDAOTN WLG ew 19 M17 M mph FM tamh 11gt 32 3an SkFe39 79 W mm A mm kmun w SzDEB hm lt 1205wa WWW 1195 4224 Mm MM 0 ex Mi H941 gawk ns rm1 91aSA L 155m gm W ww mw W 32 6 WMva w 9 VLWW aumemwpu awnmwj 5 W o gamma 1511 a 3957 W mam qW Wear m9 MW 4 A n d t 39 gt WS M wWWaF Macaw A z NsNoe Atr 90 quot V MOMMULCAM 9 F v EQPC39L ME WW9 Torrs GEquot 8me l I 1 7 3 35500 Gamma mm as We W W VW 52 55 23 2H 3H wavimw MSW FAWM Qmss w f KOtOQWIhw LEM EtvMULQWLS 4 C s45 e 17mm mm Conn l Wham fh mm 1s Emu To How 111 vo s39I H39 HA 4 O 9m W 3 A WMI39J EgCM PgtA L new KatNi L 4Wo t ew Qcmvwm m W117 meu L M


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