PurificationofAntiserumorAscitesbyProteinA/GChromatography

RequiredMaterialsandEquipment

• ProteinAorGagarosegelcolumn(10mlor5mlofpackedbeads;seeguidelinesbelowforchoiceofproteinAorproteinG)
• Ice-coldTris-bufferedsaline(TBS).Seerecipebelow.
• 5%sodiumazidesolution
• NeutralizationBuffer(NB).Seerecipebelow.
• ElutionBuffers(pH2.7andpH1.9).Seerecipesbelow.
• Centrifugetubesandmicrocentrifugetubes
• Preparativecentrifugeandmicrocentrifuge
• pHstrips
• Microtiterplatereaderwith600nmfilter
• GlassColumns

GuidelinesforChoosingProteinAAgaroseorProteinGAgarose

AntibodiesbindwithdifferentaffinitiestoproteinAandproteinGconjugatedagaroses.Usethechartbelowtochoosethebestaffinityagaroseforyourantibody

BufferPreparation

Toprepare1literofTBS(50mMTris-HCl,pH7.4;150mMNaCl;0.05%sodiumazide)addthefollowingto800mlofdistilledwater:

• 6.06gofTrisbase
• 8.77gofNaCl
• 10mlof5%sodiumazidestocksolution(Donotaddiftheantibodywillusedinassaysofcellularresponseorfunction.)

MixwellandadjustthepHto7.4with5NHCl.Bringthevolumeupto1LwithdistilledwaterandrecheckthepHafterchillingonice.

Toprepare100mlofNB(1MTris-HCl,pH8.0;1.5MNaCl;1mMEDTA;0.5%sodiumazide),addthefollowingto80mlofdistilledwater:

• 12.1gofTrisbase
• 8.7gofNaCl
• 200microlitersof0.5MEDTA
• 10mlof5%sodiumazidestocksolution(Donotaddiftheantibodywillusedinassaysofcellularresponseorfunction.)

MixthoroughlyandadjustthepHto8.0with5NHCl.Adddistilledwatertoobtainafinalvolumeof100ml.TheNaClandEDTAareaddedtopreventaggregationoftheantibodiesandlossofBIOLOGicalactivity.

Toprepare100mlofElutionBufferpH2.7(50mMGlycine-HCl,pH2.7),addthefollowingto80mlofdistilledwater:

• 0.38gofGlycine

MixthoroughlyandadjustthepHto2.7with5NHCl.Adddistilledwatertoafinalvolumeof100ml.

Toprepare100mlofElutionBufferpH1.9(50mMGlycine-HCl,pH1.9),addthefollowingto80mlofdistilledwater:

• 0.38gofGlycine

MixthoroughlyandadjustthepHto1.9with5NHCl.Adddistilledwatertoafinalvolumeof100ml.

Procedure

SectionI:PreparationofaProteinAAgaroseorProteinGAgaroseAffinityColumn

Typically,columnscontaining5mlor10mlofpackedproteinA/GAgaroseareprepared.ThesizeofthecolumnisdeterminedbythebindingcapacityofproteinA/Gandtheamountofantiserumthatmustbeprocessed.ProteinAandproteinGbindabout20mgofIgGpermlofconjugatedagarose.Therefore,thebindingcapacityofa10ml(5ml)columnis200mg(100mg)ofIgG.Ahigh-titerrabbitantiserumhasroughly5mg/mlofIgG,mouseasciteshasroughly10mg/mlofIgG,andgoatorsheepantiserumhasroughly20mg/mlofIgG.Usetheguidelinesbelowtochooseacolumnsizetoavoidexceedingthecapacityofthecolumn.Donotexceed90%ofthebindingcapacityofthecolumn.

10mlBeadVolumeProteinA/GAffinityColumn

5mlBeadVolumeProteinA/GAffinityColumn

PouringtheProteinA/GAffinityColumn

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.

SectionII.PreparationofAntiserumorAscitesforAffinityChromatography

Throughoutthissection,"antiserum"referstoantiserumorascites.

1.Thawtheantiseruminicewaterortherefrigeratorovernighttopreventaggregationofproteins.Anyproteinaggregatethatformsduringthawingmaybedissolvedbybrieflywarmingthethawedantiserumto37.

2.Addsodiumazidetoafinalconcentrationof0.05%,whichisa1:100dilutionofa5%stocksolution.

CAUTION:Sincesodiumazideistoxic,wearglovesandhandlethestocksolutionwithcare.

3.Clarifytheantiserumbycentrifugationat15,000xgfor5minutesat4.Thisstepisperformedtosedimentaggregatesofdenaturedproteinandlipid;itisanimportantstepbecauseitremovesmaterialthatcanfoulandblockthecolumn.

4.Removetheclarifiedantiserumthatliesbetweenthefloatinglipidandthepellet.Additionalfiltrationmayberequiredtoremoveresiduallipid.

SectionIII.AffinityChromatographyUsingProteinA/GAgarose

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.Storethepurifiedantibodyat2-8

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