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Second Test Material

by: Jaleesa Holmes

Second Test Material CHEM 431

Jaleesa Holmes
GPA 3.13
Biochemistry I
Dr. Inlow

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This is a study guide that is broken into sections. It gives great descriptions!
Biochemistry I
Dr. Inlow
Study Guide
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This 9 page Study Guide was uploaded by Jaleesa Holmes on Sunday October 4, 2015. The Study Guide belongs to CHEM 431 at Indiana State University taught by Dr. Inlow in Summer 2015. Since its upload, it has received 32 views. For similar materials see Biochemistry I in Chemistry at Indiana State University.


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Date Created: 10/04/15
Chem Study Guide Segment 1 Protein Methods Steps in Protein Purification 1 Obtain a source of the protein 0 Ex microbial cells plant or animal tissue 2 Homogenize the sample 0 Put the tissue sample into a blender with some buffer at an appropriate pH 3 Break open the cells of the tissue to release the protein of interest from within the cells 0 French Press Place homogenized tissue inside a chamber A piston will press down increasing the pressure inside the chamber The liquid will be forced through a very small opening The cell membrane will burst This will result in a crude extract thick sticky suspension containing particles of the broken cell membrane 0 Sonicator Place homogenized tissue into a small beaker A probe which emits ultrasound waves will be dipped into the tissue sample in the beaker The ultrasound waves will cause the cell membrane to break apart This will result in a crude extract 4 Isolate subcellular fractions or specific organelles 0 Also called subcellular fractionation 0 Separate the contents of the cells so that a certain organelle can be studied individually 0 How centrifugation works The crude extract is placed into centrifuge tubes The tubes will be placed in a rotor The rotor is placed in the centrifuge and spun at very high speeds Particles will sediment out and form a pellet at the bottom of the tube The remaining liquid about the pellet is called the supernatant 0 Differential Centrifugation Particles of different densities sediment at different centrifuge speeds Cells are broken open tissue homogenization Organelles suspended in liquid Largest particles will sediment Pour off liquid into another tube save the pellet the pellet contains whole cells nuclei cytoskeletons and plasma membranes 5 Medium particles will sediment next pellet contains mitochondria lysosomes and peroxisomes Smallest particles will sediment pellet contains ribosomes and large macromolecules Preparative Density Gradient Ultracentrifugation Usually done at higher speeds than differential centrifugation The crude extract is placed in centrifuge tubes which contains a special solution that has a density gradient the solution is denser at the bottom of the tube and less dense at the top of the tube Density gradient ultracentrifugation has greater resolving power than simple differential centrifugation Particles are separated based mainly on their differing molecular masses During centrifugation particles begin to sediment toward the bottom of the tube They eventually stop moving toward the bottom and remain suspended at the position in the tube where the density of the solution is equal to the density of the particle Due to the different densities masses of the particles different particles stop moving at different positions in the tube The solution is then drained from the bottom of the tube a layer at a time Fractionation To separate the various proteins The protein still contains impurities many other proteins present along with the protein The next step is to use a crude fractionation method Crude referring to a method that is not going to completely purify your protein Next use a more precise fractionation method to get rid of the last remaining impurities Fractionation by ammonium sulfate NH4ZSO4 precipitation The theory Proteins are said to be salted out of solution by high salt concentrations High salt concentration in the solution reduces a protein s solubility and it precipitates from the solution By adding increasing amounts of a salt in stages the proteins can be salted out at different stages which allow the proteins to be separated This process is considered crude fractionation method because it is unlikely that you ll be able to separate each individual protein Ammonium sulfate is the salt of choice because it has very high water solubility The method Protein precipitates from solution when the solution is between 35 and 50 saturated with ammonium sulfate Take the crude protein solution containing your protein and many others and add ammonium sulfate until the solution is 35 saturated This will cause many of the other proteins to precipitate but your protein won t yet precipitate 2 Centrifuge the solution so that the precipitated proteins settle out forming a pellet at the bottom of the centrifuge tube 3 Save the supernatant the liquid which still contains your protein dissolved and discard the pellet which contains other proteins 4 Take the supernatant solution and add ammonium sulfate to reach 50 saturation Your protein will precipitate from solution as the ammonium sulfate is increasing from 35 to 50 Of course some other proteins impurities will probably precipitate over this range too 5 Centrifuge the solution so that the precipitated proteins your protein as well as some others form a pellet at the bottom of the centrifuge tube 6 Pour off and discard the supernatant which contains other proteins still dissolved but save the pellet which contains your protein and some others 7 Take the pellet and dissolve it in buffer so that all proteins in the pellet are again dissolved then use a technique to remove ammonium sulfate from the solution At this stage you will have a buffered solution containing your protein along with some impurities but you will have removed many of the impurities This will make the next step in purification easier 6 Further fractionation 0 Use one or more fractionation methods to remove all remaining impurities from your protein 0 Fractionation by column chromatography Mobile phase a buffered aqueous solution Stationary Phase a porous solid material inside the column with appropriate chemical propertieslooks like fine sand A sample containing a mixture of proteins is placed at the top of the column and then the buffer is pumped into the column The proteins dissolve in the buffer and are carried down the column by the buffer as it ows through the stationary phase Different proteins migrate down the column at different rates depending on their interaction with the stationary phase Some proteins stick to the stationary phase so they travel slowly others don t interact with the stationary phase so they travel quickly The buffer that drips out of the column is collected in small increments called fractions 0 Types of column chromatography HPLC High Performance Liquid Chromatography the solventbuffer is pumped through the column under high pressure This achieves better resolution of proteins than just allowing the solvent to drip through by gravity Usually several types of chromatography must be used sequentially to remove all impurities from a protein For example start with anion exchange chromatography to get rid of some impurities then use size exclusion chromatography to get rid of remaining impurities 1 Ion Exchange Chromatography Separates proteins based on differences in net charge at a given pH 0 Cation Exchange Chromat02raphv The stationary phase is composed of microscopic polymer beads with negatively charged functional groups attached to the beads Any proteins that have a net positive charge will be attracted to the functional groups on the beads so they will travel slowly down the column so these proteins stick to the column It will take a lot of buffer to eventually wash them off the column Proteins with a large net positive charge may never come off the column under these conditions Any proteins that have a net negative charge will not be attracted to the functional groups of the stationary phase so they will be carried quickly down the column by the buffer These proteins are interacting strongly with the negative functional groups on the column To get them off the column something must be done to decrease the strength of their interaction with the stationary phase You could begin to put a buffer of higher pH through the column this will deprotonate some of the functional groups on the proteins so that their net positive charge decreases and they don t interact as strongly with the stationary phase If the pH change is drastic enough the proteins may even become negatively charged You would continue using this higher pH buffer until the proteins elute from the column Anion Exchange Chromat02raphv The surfaces of the beads are covered with positively charged functional groups Proteins with net negative charge are attracted to the positive functional groups and travel slowly down the column stick to the column they exchange places with the Cl counter ions Proteins with net positive charge are not attracted to stationary phase and are carried quickly down the column by the buffer O The particular resin represented here is called DEAE resin Di Ethyl Amino Ethyl It is one of the most commonly used resins for protein purification 2 Size Exclusion Chromatography O O The stationary phase is composed of microscopic cross linked polymer beads that have pores of a specific size range Small proteins will fit into the pores so they will meander in and out of the pores as they are carried down the column by the buffer Hence they travel slowly and take a long time to come off the column Large proteins will not be able to fit into the pores so they only move between the beads and don t meander in and out of the pores They take a direct path down the column They travel quickly down the column Proteins are separated by this method based on their different sizes The shape of a protein will also affect the amount of time it takes to elute 3 Affinity Chromatography O O The stationary phase is composed of polymer beads with a ligand cross linked to the beads Any proteins that bind to the ligand will stick to the stationary phase and will not elute from the column Other proteins will be washed off the column quickly in the buffer In order to make the bound proteins elute from the column a solution containing the ligand can be used The ligand in this solution competes for the binding of the protein molecules As this solution ows through the column the protein molecules dissociate from the column bound ligand and instead bind to the free ligand in the solution allowing these proteins to be washed off the column Overall proteins that contain tryptophan andor tyrosine residues absorb light at 280 nm You can monitor the absorbance of 280 nm light for the solution that comes off the column Pure buffer won t absorb 280 nm light Therefore if the absorbance of the solution coming off the column is high it must contain protein Ultraviolet light spectrophotometer for measuring the absorbance of 280 nm light A protein sample dissolved in buffer is placed in a cuvette and the cuvette is placed in the spectrophotometer where it is eXposed to 280 nm ultraviolet light The machine gives a read out of the absorbance A280 8C1 A280 absorbance of 280 nm light 8 molar extinction coefficient Lmol lcm l it is different for different proteins and depends on the number depends on the number of Tyr and Trp residues in the protein c concentration moll l path length of the light absorbing sample cm 7 Analysis of the purified protein Once the protein of interest has been satisfactorily purified it can be analyzed Common analyses might include enzyme assays binding assays mass spectrometry NMR uorescence spectroscopy amino acid sequence determination or electrophoresis Electrophoresis the migration of charged proteins in an electric field Used to purify small amounts of protein You have a container containing a buffer in the container is a gel that is submerged in the buffer A gel is a cross linked web made of polymers it is a thin sheet of oppy gelatinous material that is essentially colorless kind of like unflavored gelatin An electric field is applied to the gel At one end of the gel is the anode and at the other end is the cathode You put a protein sample on the gel As long as the electric field is being applied the protein will move through the gel in one direction or the other toward the anode or cathode depending on its net charge Types 1 sodium dodecvl sulfate Dolvacrvlamide gel electrophoresis SDS PAGE proteins migrate in an electric field according to molecular weight Proteins migrate through the gel at different speeds based on differences in size molecular weight The buffer that surrounds the gel and that is mixed with the protein sample always contains SDS 0 SDS is an amphipathic molecule and is a detergent 0 SDS binds to proteins about 1 SDS molecule binds for every 2 amino acid residues in a protein imagine a protein molecule coated in SDS molecules 0 Since SDS is negatively charged the resulting proteinSDS aggregate will have a large net negative charge The large amount of bound SDS swamps any net charge whether it was negative or positive that the protein may have had on its own 0 Steps I 1 SDS molecules bind to the protein molecules so all proteins have a large net negative charge 2 Protein samples placed on the gel at the end near the cathode lt gt I 3 An electric field is applied and the proteins migrate through the microscopic holes in the web like meshwork of the gel They all move toward the anode because they are all negatively charged They move in a path straight downward so the 4 samples shown in the diagram will not end up miXing with one another I 4 Small proteins move through the gel quickly because they fit through the holes relatively easily large proteins move slowly because they get caught in the web more easily I 5 When the fastest moving proteins have gotten close to the bottom of the gel the electric field is removed so everything stops moving The small proteins will be located near the bottom anode end of the gel while larger proteins will be closer to the top cathode end They have been separated according to their molecular weights sizes I 6 The gel is removed from the apparatus It is soaked in a dye which stains only the proteins so that it is possible to see their locations I 7 A protein s molecular weight can be calculated based on how far it moved down the gel Used to monitor the progress of protein purification Seeing only one protein band on a gel does not necessarily mean that only one protein is present The band could consist of two or more proteins with the same molecular weight If you see only one band and think your protein is pure you need to use some other method to confirm that only one protein is present 2 Isoelectric Focusing IEF proteins migrate in an electric field containing a pH gradient according to pl 0 O O The gel contains an ampholyte solution Ampholyte an amphoteric electrolyte a molecule that can act as an acid or base it has carboxyl and amino groups The presence of ampholytes in the gel sets up a pH gradient in the gel after an electric field is applied Therefore one end of the gel has a higher pH while the other end has a lower pH The pH could range from 3 to 10 or from 4 to 6 depending on the particular ampholytes used 0 When an electric field is applied proteins migrate through the gel until they reach an equilibrium position where they stop moving The position where a protein stops is determined by its pl 0 The pI of the protein is the pH where the protein s net charge is zero Therefore different proteins can be separated by isoelectric focusing based on their different pI s 0 Steps I 1 An ampholyte solution is incorporated into the gel An electric field is applied which sets up a stable pH gradient in the gel I 2 Protein samples are placed on the gel at the cathode end where the pH will be highest All the proteins will have a net negative charge in this high pH environment because most functional groups will be deprotonated at this high pH I 3 An electric field is again applied and the proteins migrate through the gel They all move down toward the anode because they are all negatively charged initially As they move downward they are moving into a lower pH environment due to the pH gradient in the gel I As the pH decreases the H increases Therefore functional groups on the protein will be protonated when the pH of the environment is approximately equal to the pKa of the functional group I As the protein keeps moving downward into a lower pH environment eventually the net charge of the protein becomes zeroThis means that the pH of the environment is equal to the pI of the protein I Since the protein has a net charge of zero it is no longer attracted to either the anode or the cathode so it stops moving and stays at that position Different proteins will stop moving at different positions in the gel because different proteins have different combinations of functional groups and different pI s I They are separated by this technique according to their pI s I 4 When all the proteins have reached equilibrium and stopped moving the electric field is removed The gel is then removed from the apparatus and soaked in a dye to stain the proteins so they can be seen It is unlikely that two proteins will have the same molecular weight AND the same pI If only one band is present on the IEF gel it can be reasonable that only one protein is present Determination of the amino acid sequence of a protein Edman degradation amino terminal sequencing 0 A machine called a sequenator is used to carry out this chemical reaction An efficient sequenator can determine the first 50 residues of a protein 0 This method allows the determination of the sequence of amino acids that make up a protein by removing one amino acid at a time from the N terminus and identifying it 0 Used to confirm the identity of a protein 0 Steps 1 Break any disulfide bonds present in the protein 2 Break the protein into small fragments each 50 amino acids or less 0 This is done by miXing the protein with a protease an enzyme that catalyzes the hydrolysis of peptide bonds essentially breaking the protein into smaller fragments O Proteases are specific for certain peptide bonds They only cleave peptide bonds next to 39 c amino acid residues O EX CNBr is just a chemical reagent not a protease Protease cleaves on C terminal side of this residue 3 Sequence each fragment using Edman degradation 0 Proteolytic fragments fragments produced by action of a protease 4 Determine the order in which the fragments occur in the intact protein 0 Determine the order in which the fragments occur in the intact protein 0 Repeat step 2 using a new sample of protein and cleaving it with a different protease Then you can compare the fragments obtained using two different proteases and hopefully determine their order in the protein 0 A 3rd or 4th cleavage method may be necessary Protein sequences can be determined indirectly by sequencing the gene that encodes the protein DNA sequencing can be faster and more accurate than protein sequencing


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