MCB 450 1st Edition Lecture 5 Outline of Last Lecture I Tertiary and Quaternary Structure of Proteins a Globular Proteins b Motifs and Domains c Fibrous Proteins d Collagen II Protein Bioinformatics III Protein Folding Denaturation IV Post translational Modifications of Proteins V Amyloidoses Outline of Current Lecture I Tissue Cell Fractionation a Centrifugation II Protein Purification a Chromatography b Electrophoresis III Antibodies IV Peptide fragmentation sequencing a Edman Degradation V Analysis of Protein Structure by X ray Crystallography Current Lecture Why purify proteins Often purify proteins to create medicines Tissue cell disruption There are many ways and strategies to purify proteins o Could use ultrasound sonication These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute Different cell components can be separated by centrifugation Centrifugation exploit the fact that different cellular components have different densities At relatively low speed pellet contains nuclear fraction Medium speed pellet mitochondrial fraction High speed pellet microsomal fraction Differential centrifugation because we are using different speeds Density gradient centrifugation Density gradient centrifugation mixture of low density and high density solution the component that you are testing will go to the portion of the tube that is equal to is density during centrifugation What does S mean Svedberg Units Sedimentation Coefficient The higher the sedimentation coefficient the faster the component will move in the centrifuge and thus the denser the material is Low sedimentation coefficient slower movement less dense Protein purification If protein has never been separated before then you will have to try a few different strategies trial and error Differences in protein solubility as a function of salt concentration If you add salt then solubility increases Separation by size dialysis Dialysis relies on having a semipermeable membrane Small molecules will move to areas of lower concentration but larger molecules will not move Kidney dialysis machines do the same thing they remove excess salt from the kidneys and bloodstream Protein purification by chromatography Chromatography based on motilities through some matrix Separation by size gel filtration chromatography Gel filtration separating on the basis of size o Liquid acts as a sieve o Large molecules with move through more quickly allowing separation o If your protein is small enough to enter the carbohydrate polymer bead then the sample will take much longer to separate out of the solution Separation by charge ion exchange chromatography Solvent pH affects charge of protein depending on pH Compare pI of protein to the solvent pH picture As pH drops below pI protein will be more and more protonated and positively charged pKa of buffer pKa of the resin and the isoelectric point of your protein are all very important Cation exchanger will bind to cations and anion exchanger will bind to anions Cation exchangers Cation exchanger will work when the pKa of the resin is less than the pH and then the pH has to be less than the pI of the protein Anion exchangers Anion exchanger will work when the pI of the protein is less than the pH and then the pH is less than the pKa of the resin Ion exchange chromatography on a cation exchanger Positive charged molecules will interact with the resin and then the negatively charged molecules will flow through Getting proteins off the cation exchanger All of these interactions are reversible Displaying proteins by SDS polyacrylamide gel electrophoresis First denature the protein in a detergent SDS when added to a protein and will bind to the protein and denature it o Then confers a large negative charge on the protein o So when we separate the proteins in an electric field they will be separated on weight because they will all have a negative charge Use essentially a molecular sieve to separate them based on molecular weight o Polyacrylamide gel Proteins are loaded at the top in different well Then the electric field is applied and the molecules will be attracted to the positive electrode Lighter molecules will move faster and the heavier molecules will move slower Detect the proteins by staining Proteins separated by SDS PAGE can be visualized by staining gel Standards is not in a linear scale it is in a log scale Isoelectric focusing to separate proteins according to pI in gel containing a stationary pH gradient Gel with pH gradient made in advance gel vs porous so proteins migrate freely Isoelectric focusing will separate based on isoelectric point not based on molecular weight o With non denatured proteins 2 D gels If you combine isoelectric focusing and SDS electrophoresis you get 2 D gels Shows all proteins made in E coli Monitoring protein purification by SDS PAGE Once you have a single protein you can create other reagents with it o Create antibodies Two types of antibodies can be generated Polycolonal injected into rabbit for example the rabbit will create antibodies to your protein o Mixture of individual antibody molecules each specific for different parts epitopes of the antigen molecule Monoclonal get clone of a antibody that will create only one antibody Immunoprecipitation Antibody to this protein is bound to some insoluble bead we place these beads in the solution and centrifuge ELISA ELISA way of looking for antibodies to an interesting protein Immunofluorescence Immunofluorescence actin or vinculin are attached to fluorescent groups Separation of derivatized amino acids on an ion exchange column Washing by increasing pH This determines the composition Determination of peptide and protein sequences Edman degradation essentially removing one amino acid at a time to determine sequencing Edman degradation First round first peptide bond is cleaved and none of the others Each one will differ on account of the R chain There are machines that do the Edman degradation procedure