蛋白质的色谱分离3(英文).ppt

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1、HydrophobicChromatography(色谱法,层析法)IonExchangeGelfiltration/SizeexclusionAffinityThemostimportantseparatestepAffinityChromatography（亲和层析）Affinitychromatographyseparatesproteinsonthebasisofareversibleinteractionbetweenaprotein(orgroupofproteins)andaspecificligandcoupledtoachromatographymatrix.The

2、techniqueoffershighselectivity,hencehighresolution,andusuallyhighcapacityfortheprotein(s)ofinterest.Affinitychromatographyisuniqueinpurificationtechnologysinceitistheonlytechniquethatenablesthepurificationofabiomoleculeonthebasisofitsbiologicalfunctionorindividualchemicalstructure.Purificationt

3、hatwouldotherwisebetime-consuming,difficultorevenimpossibleusingothertechniquescanoftenbeeasilyachievedwithaffinitychromatography.AffinityChromatographySometypicalbiologicalinteractions,frequentlyusedinaffinitychromatography:Enzymesubstrateanalogue,inhibitor,cofactor.Antibodyantigen,virus,cel

4、l.Lectinpolysaccharide,glycoprotein,cellsurfacereceptor,cell.Nucleicacidcomplementarybasesequence,histones,nucleicacidpolymerase,nucleicacidbindingprotein.Hormone,vitaminreceptor,carrierprotein.Glutathioneglutathione-S-transferaseorGSTfusionproteins.MetalionsPoly(His)fusionproteins,nativep

5、roteinswithhistidine,cysteineand/ortryptophanresiduesontheirsurfaces.Theprocessofusingaffinitychromatography:Theprocessofusingaffinitychromatography:3.Targetproteinisrecoveredbychangingconditionstofavorelutionoftheboundmolecules.Elutionisperformedspecifically,usingacompetitiveligand,ornon-speci

6、fically,bychangingthepH,ionicstrengthorpolarity.Targetproteiniscollectedinapurified,concentredform.4.Affinitymediumisre-equilibratedwithbindingbuffer.TheElutionofaffinitychromatography:Thereisnogenerallyapplicableelutionschemeforallaffinitymedia.Referencetomanufacturer’sinstructions,thescientif

7、icliteratureandafewsimplerulesshouldresultinaneffectiveelutionmethodthatelutesthetargetproteininaconcentratedform.Elutionmethodsmaybeeitherselectiveornon-selective.Anaffinitychromatogram:Troubleshooting:Troubleshooting:Troubleshoo