MCB 450 1st Edition Lecture 5Outline of Last Lecture I. Tertiary and Quaternary Structure of Proteinsa. Globular Proteinsb. Motifs and Domainsc. Fibrous Proteinsd. CollagenII. Protein BioinformaticsIII. Protein Folding & DenaturationIV. Post-translational Modifications of ProteinsV. AmyloidosesOutline of Current Lecture I. Tissue & Cell Fractionationa. CentrifugationII. Protein Purificationa. Chromatographyb. ElectrophoresisIII. AntibodiesIV. Peptide fragmentation & sequencinga. Edman DegradationV. Analysis of Protein Structure by X-ray CrystallographyCurrent LectureWhy purify proteins?-Often purify proteins to create medicinesTissue & cell disruption-There are many ways and strategies to purify proteinsoCould use ultrasound -- sonicationThese 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 speedsDensity 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 centrifugationWhat 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 denseProtein 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 bloodstreamProtein purification by chromatography-Chromatography based on motilities through some matrixSeparation by size: gel filtration chromatography-Gel filtration -- separating on the basis of sizeoLiquid acts as a sieveoLarge molecules with move through more quickly allowing separationoIf your protein is small enough to enter the carbohydrate polymer bead then the sample will take much longer to separate out of the solutionSeparation 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 anionsCation 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 proteinAnion 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 resinIon exchange chromatography on a cation exchanger-Positive charged molecules will interact with the resin and then the negatively charged molecules will flow throughGetting proteins off the cation exchanger-All of these interactions are reversibleDisplaying 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 itoThen confers a large negative charge on the proteinoSo 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 weightoPolyacrylamide 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 stainingProteins separated by SDS-PAGE can be visualized by staining gel-Standards -- is not in a linear scale, it is in a log scaleIsoelectric 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 weightoWith non-denatured proteins2-D gels-If you combine isoelectric focusing and SDS electrophoresis you get 2-D gels-Shows all proteins made in E. coliMonitoring protein purification by SDS-PAGE-Once you have a single protein you can create other reagents with itoCreate antibodiesTwo types of antibodies can be generated-Polycolonal -- injected into rabbit for example the rabbit will create antibodies to your protein oMixture 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 antibodyImmunoprecipitation-Antibody to this protein is bound to some insoluble bead we place these beads in the solution and centrifugeELISA-ELISA -- way of looking for antibodies to an interesting proteinImmunofluorescence-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 compositionDetermination of peptide and protein sequences-Edman degradation -- essentially removing one amino acid at a time to determine sequencingEdman 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