PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 605-1MCB 450Lecture 5Techniques in Protein BiochemistryTissue & Cell FractionationCentrifugationProtein PurificationChromatographyElectrophoresisAntibodiesPeptide fragmentation & sequencingEdman DegradationAnalysis of Protein Structure by X-ray CrystallographyWhy purify proteins?5-2•Study their enzymatic activity and what factors affect activity•Determine their structures (e.g. by X-ray diffraction) sowe can relate structure to function•Find compounds that block enzyme activity = medicines•Raise antibodies to them, so we can detect the proteinin living cells•Use enzymes therapeutically & industriallyChallenge:Need to get our protein out of the tissues / cells that contain it,and separate it from all other proteinsSonication: break cellsw. high frequency soundTissuehomogenate/cell suspensionBacterialcellsGrind in mortar & pestlew. sand/alumina/liq N2Shear cells betweenclose-fitting plunger &wall of thick tubeForce cells throughsmall hole usinghigh pressurehttp://cellbiologyolm.stevegallik.org/node/74Tissue & celldisruption5-3Exploits the fact that differentcellular components havedifferent densities: sediment("pellet") at different g forcesDifferent cell components can be separatedby centrifugation5-4Spin at successivelyhigher g-forces(faster speeds)DifferentialcentrifugationDensity gradient centrifugation5-5Separates protein complexes and different cellular membranesDuring centrifugation at high g,component migrates down tubeto region of equal density LESSDENSEMOREDENSEe.g. sucrosesolutionsDensity gradient centrifugation5-6LESSDENSEMOREDENSECollection of different fractions"S" depends on…particle shape, size, density & massdensity of the solution.The smaller the S, the more s l o w l y the particle / molecule moves in a centrifugal field.What does "S" mean?"Svedberg" Units, Sedimentation Coefficient5-7The functional ribosome is 70S. It is composed ofone 30S subunit and one 50S subunitSolubility Increasing ionic strength at first increases solubility of proteins (salting in), then decreases it (salting out)Size/shape Dialysis, size-exclusion chromatography(gel filtration)Charge (pI) Ion exchange chromatography, electrophoresisBinding to small molecules Affinity chromatography(small molecule immobilized)Protein purification5-8Protein property MethodFor most protein purifications, the steps are carried out at ~ 4°Cto slow down degradation (crude samples contain proteases).Goal: Separate your protein from all others in a cell extract Strategy: Use several different procedures:exact procedures, & order in which they are carried out,differ from protein to protein.Procedures exploit characteristic properties of the proteinDifferences in protein solubility as a function ofsalt concentration5-9Increasing ionic strength at firstincreases protein solubilityAt high salt concs.,competition betweenadded salt ions anddissolved proteinfor water molecules.Hydration shell isstripped off protein,and it precipitatesSeparation by size: dialysisCan use dialysis to remove excess saltfrom a protein solution, or to change thebuffer a protein is in.5-10Protein purification by chromatography5-11•Principle of chromatography:Separation of molecules based on theirrelative mobilities through a matrixSeparation of molecules is increasedby retarding their relative mobilitiesby increasing their interactions with the matrix•Examples:Gel filtration: mobility differences due to sizeIon exchange: mobility differences due todifferences in electrostatic attraction to matrixAffinity chromatography: immobilization due tobinding to a specific ligandSeparation by size: gel filtration chromatogaphyLiquid chromatography in a “column” is the mostwidely used method for separating proteins A buffer the proteincan dissolve incollect fractions5-12Beads for gel filtration5-13• Often high mol. wt. polymers of glucose (“Sephadex”)• Pore size in beads can be varied• Exclude proteins above a certain mol. wt.,which then pass down column more quickly• Can calibrate column with standard proteins ofknown mol. wt. & estimate size of unknownSeparation by charge: ion exchange chromatographyCompare pI of protein to the solvent pH:if pH > pI, protein will be -vely chargedif pH < pI, protein will be +vely chargedRELATIVELY UN-PROTONATEDRELATIVELY PROTONATED5-14Pass protein mixture through a column of ion exchanger (“resin”)= polymer containing bound charged groupsCation exchangerLow pKa groups,binds +vely charged proteinsHigh pKa group,binds -vely charged proteinsAnion exchanger-+5-15 CATION EXCHANGERSMOST EFFECTIVE WHEN pKa(resin) < pH < pI(protein)So protein+vely charged(protonated)So resin –vely charged(deprotonated)MOST EFFECTIVE WHEN pI(protein) < pH < pKa (resin)So protein-vely charged(deprotonated)So resin+vely charged(protonated)5-16 ANION EXCHANGERSIon exchange chromatography on a cation exchanger5-17pH of buffer > pKa of resin,so resin –vely chargedpH of buffer < pI of protein,so protein +vely chargedGetting proteins off the cation exchangerChange the pHIn this case, raising pHwill remove +ve chargefrom the protein5-18increase the ionic strengthNa+ ions will compete with+vely charged groups on a proteinfor binding to a cation exchanger.Separation by binding specificity:affinity chromatography5-19•Exploits the fact that some proteins bind very tightly to a specific ligand•Immobilize the ligand on a column- Binds to most proteins in amounts ~ proportional to mol. wt. of the protein (~ 1 SDS / 2 ).- Bound SDS contributes large -ve charge, >> intrinsic charge of protein,so proteins are separated almost exclusively on the basis of mol. wt.- Protein samples are denatured by heating in SDS + mercaptoethanol to reduce S-S bonds: = “denaturing electrophoresis”Displaying proteins bySDS polyacrylamide gel electrophoresis (SDS-PAGE)-15-20Displaying proteins bySDS polyacrylamide gel electrophoresis (SDS-PAGE)-25-21Samples of denatured proteins loaded at top ofpolyacrylamide gel held between two glass
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