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BIOC23H3 (23)
Lecture

BIOC23: Lec 8 - Affinity Chromatography.doc

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Department
Biological Sciences
Course
BIOC23H3
Professor
Rongmin Zhao
Semester
Winter

Description
Lec 8 Affinity Chromatographyspecialized adsorption chromatography specific physical binding between 2 solutesthe matrixanchored ligand can besubstratecofactorcompetitive inhibitorantibodyto purify antigenexpensive and nonspecific binding effectsMatrixlarge pore sizeoActivated sepharosecrosslinked agaroseoPolyacrylamide BiogeloGlassopolyvinylLigand must bind target withoHigh affinityoReversibleoThis means low equilibrium dissociation constant smaller means stronger binding oFastonfast offPutting ligand on the matrixoCan be covalentnoncovalentoPeptideprotein ligandusually covalent oPreactivated mediaCnBr activated agarosesepharoseAfter activation beads can react with ligandNaHCO3good bufferslightly basicNot all affinity chromatography needs covalent attachment for some metal ionsuse chelatorsSmall ligands less than 1000 daltonsneed spacer to couple the ligand to matrixoRisk of small ligandsSteric interference btn matrix and target mocLow accessibility of ligandTherefore use spacer armUsu hydrophilicoAgarose matrix beadspacer armsmall ligandbind interest proteinLigands used for GROUPSPECIFIC isolate more than one targetoProtein A bind antibody at Fc fragmentcommon to several antibodiesoProtein G bind antibody at Fc fragmentoTransition metal ions to bind proteinspeptides which contain accessible Hiscontains imidazole R group which has high affinity for certain metal ionsoConcanavalin Abinds sugar
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