抗体纯化技术

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1、AntibodyPurification王雷20100702抗体纯化样品准备多步纯化一步纯化抗体一般性质了解Antibodystructure,commonstructure：twoidenticalheavy(H)chainstwoidentical,non-glycosylatedlight(L)chainsAntibodyclassificationaccordingtotheirHchaincomponents:IgG(g),IgA(a),IgM(μ),IgD(d)andIgEtwotypesoflightchain,kandl.Physic

2、o-chemicalpropertiesofmouseimmunoglobulinsSourcesandtheirassociatedcontaminantsSamplepreparationCentrifugationFiltrationBufferexchangedesaltingCentrifugation：removeslipidsandparticulatematter,suchascelldebrissmallsamplevolumesorproteins10000xg15minutescelllysates40000–50000xg30

3、minutesFiltration：FiltrationremovesparticulatematterandissuitableforsmallsamplevolumesNominalporesizeoffilterParticlesizeofchromatographicmedium1μm90μmandupwards0.45μm34μm0.22μm3,10,15μmorwhenextracleansamplesorsterilefiltrationisrequiredSamplepreparationbeforepurificationRemov

4、alofspecificimpurities,suchaslipoproteinsorphenolred,fromthesourcematerialRemovalofgrossimpurities,suchasbulkprotein,fromthesourcematerial.Bufferexchangeanddesaltingtotransfersampletothecorrectbufferconditions(pHandsaltconcentration）RemovalofgrossimpuritiesPrecipitationtechniqu

5、es：differentialsolubilityconcentrationpHtemperaturePrecipitationagentCommentAmmoniumsulphate（硫酸铵）Stabilizesproteins.Helpsreducelipidcontent.Antibodyconcentrationshouldbe>1mg/ml.Samplecanbeconcentratedintoapellet.Mostsamplesretainnativeform.ExcellentifHICissubsequentpurification

6、step.Caprylicacid（辛酸）Sampleremainsinsupernatantandisnotconcentrated.Antibodyconcentrationshouldbe>1mg/ml.Polyethyleneglycol（聚乙二醇）Stabilizesproteins.FrequentlyusedforpolyclonalantibodiesandmonoclonalIgM.IgMsprecipitatemorereadilythanIgGs.PEG-600behavesasaMr50000–100000globularpr

7、oteiningelfiltrationandiseasilyseparatedfromIgM,butdifficulttoremovefromsmallermolecules.硫酸铵沉淀流程centrifugefilteradjustpH.precipitationcentrifugeremovesupernatantwashpelletdissolvepelletbufferexchange辛酸沉淀流程dilutionadjustpHaddcaprylicacidCentrifugesupernatantadjustpHremovethecapr

8、ylicfurtherpurificationdialysisdesaltingcolumnbufferex