AntibodiesbindwithdifferentaffinitiestoproteinAandproteinGconjugatedagaroses.Usethechartbelowtochoosethebestaffinityagaroseforyourantibody SpeciesandTypeofAntibody Agarose Toprepare1literofTBS(50mMTris-HCl,pH7.4;150mMNaCl;0.05%sodiumazide)addthefollowingto800mlofdistilledwater: MixwellandadjustthepHto7.4with5NHCl.Bringthevolumeupto1LwithdistilledwaterandrecheckthepHafterchillingonice. Toprepare100mlofNB(1MTris-HCl,pH8.0;1.5MNaCl;1mMEDTA;0.5%sodiumazide),addthefollowingto80mlofdistilledwater: MixthoroughlyandadjustthepHto8.0with5NHCl.Adddistilledwatertoobtainafinalvolumeof100ml.TheNaClandEDTAareaddedtopreventaggregationoftheantibodiesandlossofBIOLOGicalactivity. Toprepare100mlofElutionBufferpH2.7(50mMGlycine-HCl,pH2.7),addthefollowingto80mlofdistilledwater: MixthoroughlyandadjustthepHto2.7with5NHCl.Adddistilledwatertoafinalvolumeof100ml. Toprepare100mlofElutionBufferpH1.9(50mMGlycine-HCl,pH1.9),addthefollowingto80mlofdistilledwater: MixthoroughlyandadjustthepHto1.9with5NHCl.Adddistilledwatertoafinalvolumeof100ml. Typically,columnscontaining5mlor10mlofpackedproteinA/GAgaroseareprepared.ThesizeofthecolumnisdeterminedbythebindingcapacityofproteinA/Gandtheamountofantiserumthatmustbeprocessed.ProteinAandproteinGbindabout20mgofIgGpermlofconjugatedagarose.Therefore,thebindingcapacityofa10ml(5ml)columnis200mg(100mg)ofIgG.Ahigh-titerrabbitantiserumhasroughly5mg/mlofIgG,mouseasciteshasroughly10mg/mlofIgG,andgoatorsheepantiserumhasroughly20mg/mlofIgG.Usetheguidelinesbelowtochooseacolumnsizetoavoidexceedingthecapacityofthecolumn.Donotexceed90%ofthebindingcapacityofthecolumn. 1.Useapipettotransferthedesiredvolumeofa50%proteinA/Gagaroseslurrytoavacuumflask.Remembertotransferenoughslurrytoprepareacolumnwiththedesiredbedvolumeandcapacity. 2.Placeastopperinthevacuumflaskandapplyvacuumforatleast15minutesatroomtemperature.Thisstepremovesgasfromtheagaroseandisnecessarytopreventbubbleformationinthecolumnthatwouldreducethecolumn"scapacityandresolution. 3.Whiledegassingtheagarose,chilltheTBSonice.PrepareTBSwithoutazideforantibodiesthatwillbeusedinvivoorincellularassays.TBSisusedtoequilibrateandwashtheproteinA/Gcolumn. 4.SlowlyaddthedegassedproteinA/Gagarosetoaglasscolumn,e.g.,aBioradEconoColumn,usingawideborepipettopreventrupturingthebeads. 5.Packthecolumnataflowrateofabout1-2mlpermin.Donotletthecolumnrundry!FlowratemaybecontrolledbyusingaMariotteflaskorpump. 6.Washthecolumnwith10bedvolumesofice-coldTBS.Thisstepchillsthecolumn,whichreducesnonspecificbindingofproteinsandslowsthemetabolismofanyremainingviablemicrobes. Throughoutthissection,"antiserum"referstoantiserumorascites. 1.Thawtheantiseruminicewaterortherefrigeratorovernighttopreventaggregationofproteins.Anyproteinaggregatethatformsduringthawingmaybedissolvedbybrieflywarmingthethawedantiserumto37. 2.Addsodiumazidetoafinalconcentrationof0.05%,whichisa1:100dilutionofa5%stocksolution. CAUTION:Sincesodiumazideistoxic,wearglovesandhandlethestocksolutionwithcare. 3.Clarifytheantiserumbycentrifugationat15,000xgfor5minutesat4.Thisstepisperformedtosedimentaggregatesofdenaturedproteinandlipid;itisanimportantstepbecauseitremovesmaterialthatcanfoulandblockthecolumn. 4.Removetheclarifiedantiserumthatliesbetweenthefloatinglipidandthepellet.Additionalfiltrationmayberequiredtoremoveresiduallipid. 1.Savea500microlitersampleoftheclarifiedantiserumfortestingalongsidethepurifiedIgGbecausetheantibodymaybeacidlABIle. 2.Addtheclarifiedantiserumtothecolumnataflowrateof2mlperminute.(Calibratetheflowrateusinga15mlconicalcentrifugetubeasthereceivingvessel.Adjustthestopcockonthecolumntogiveaflowof2mlperminute.Donotexceedaflowrateof2mlperminute.)Passtheantiserumthroughthecolumntwiceandsavetheflowthroughincasetheantibodydidnotbindtothecolumn. 3.WashthecolumnwithavolumeofTBSequalto10foldthevolumeofantiserumloadedonthecolumn.Forexample,if30mlofantiserumwereloadedonthecolumn,washwith300mlofPBS.Savethewashincasetheantibodywaseluted. 4.Afterwashingthecolumn,collecta20microliterfractionandtestforproteinbyaddingitto180microlitersofCoomassieBluereagenttotestforprotein.Ifthesampleturnsblue,washthecolumnwithanadditional50mlofTBSandrepeattheanalysisforprotein. 5.Prepare1.5mlmicrocentrifugetubesforthecollectionofelutedantibody.Obtainonetubeforeachmlofantiserumplusfiveextra1.5mltubes.Add100microlitersofNeutralizationBuffer(NB)toeachtube.NotethatIgGmaybeelutedfromproteinA/Gagarosebyachangeintemperature,ionicstrength,and/orpH.AlloftheseconditionsaffectthebindingbetweenthechargedaminoacidsofproteinA/GandtheFcregionoftheIgGheavychain.Whenelutingwithacidicbuffer,theacidmustbeneutralizedasquicklyaspossIBLetopreventdenaturationoftheIgG. 6.Oncethecolumnhasbeenwashed,draintheTBSfromthecolumnbedtoavoidunnecessarydilutionoftheElutionBuffer.Donotletthecolumnstanddry. 7.GentlyaddElutionBufferpH2.7atroomtemperaturetothecolumn.Becarefulnottodisruptthecolumnbed.Use15mlofElutionBufferpH2.7iflessthan20mlofantiserumwasloaded.Use20mlofElutionBufferpH2.7if20-30mlofantiserumwasloaded.Use25mlofElutionBufferpH2.7if30-36mlofantiserumwasloaded.NotethatthesevolumesareguidelinesandElutionBuffermusebeaddeduntilallproteinhasbeenelutedfromthecolumn. 8.Collectroughly1mlfractionsinthetubespreparedinstep5above.Mixeachfractionimmediatelyandplaceonicebeforecollectingthenextfraction.NeutralizingtheacidpHpreventsdenaturationoftheantibodiesandlossofbiologicalactivity. 9.Add10mlofElutionBufferpH1.9atroomtemperaturetothecolumn;becarefulnottodisruptthecolumnbed.Collect,mix,andsavefractionsasdescribedinstep8above. 10.Removea20microlitersampleofeachfractionandaddto180microlitersofCoomassieBluereagentinamicrotiterplatetomonitorproteinelution.Continuetocollectfractionsuntilthecolordropstoorjustbelowbackground. 11.Readthemicrotiterplateat600nmandcombineallfractionswithanabsorbancegreaterthanorequalto0.2abovebackgroundat600nm.Forexample,ifthebackgroundis0.18,combineallfractionswithanabsorbancegreaterthan0.38. 12.Oncetheabsorbancehasdroppedbelow0.2,washthecolumnwith200mlofice-coldTBSwith0.05%sodiumazide. 13.Storethecolumnat2-8. 14.UsepHpapertocheckthepHofthecombinedfractions.IfthepHislessthan7.0,useNBtoadjustthepHtoapproximately7.4. 15.UsetheBradfordAssaywithIgGasastandard,todeterminetheconcentrationoftheantibodyinthecombinedfractions.Addglycerolto10%ofthetotalvolumeiftheantibodyconcentrationofthecombinedfractionsislessthan0.5mg/ml. 16.Storethepurifiedantibodyat2-8PurificationofAntiserumorAscitesbyProteinA/GChromatography
RequiredMaterialsandEquipment
GuidelinesforChoosingProteinAAgaroseorProteinGAgarose
RabbitIgG ProteinAorProteinG MouseIgG1 ProteinG MouseIgG2 ProteinAorProteinG MouseIgG3 ProteinG SheepIgG ProteinGbutbindsweakly RatIgG ProteinGbutbindsweakly GuineaPigIgG ProteinA DogIgG ProteinA GoatIgG ProteinG PigIgG ProteinA BufferPreparation
Procedure
SectionI:PreparationofaProteinAAgaroseorProteinGAgaroseAffinityColumn
10mlBeadVolumeProteinA/GAffinityColumn
SourceofAntibody Concentration VolumeforCapacity Volumefor90%Capacity RabbitAntiserum 5mg/ml 40ml 36ml MouseAscites 10mg/ml 20ml 18ml Goat/SheepAntiserum 20mg/ml 10ml 9ml 5mlBeadVolumeProteinA/GAffinityColumn
SourceofAntibody Concentration VolumeforCapacity Volumefor90%Capacity RabbitAntiserum 5mg/ml 20ml 18ml MouseAscites 10mg/ml 10ml 9ml Goat/SheepAntiserum 20mg/ml 5ml 4.5ml PouringtheProteinA/GAffinityColumn
SectionII.PreparationofAntiserumorAscitesforAffinityChromatography
SectionIII.AffinityChromatographyUsingProteinA/GAgarose