Forlabeling100µgantibody(assumingthetargetantibodyconcentrationis1mg/mL),mix5µL(5%ofthetotalreactionvolume)ofReactionBuffer(ComponentC)with100µLofthetargetantibodysolution.

Note1:Ifyouhaveadifferenceantibodyconcentration,adjusttheantibodyvolumeaccordinglytomake~100µgantibodyavailableforyourlabelingreaction.
Note2:Theantibodyshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheantibodyisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note4:TheAntibody-Buccutite™MTAreactionefficiencyissignificantlyreducediftheantibodyconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalantibodyconcentrationrangeof1-10mg/mLisrecommended.

2.1AddtheantibodysolutiondirectlyintothevialofBuccutite™MTA(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.

2.2KeeptheAntibody-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.

Note:TheAntibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.

3.1Inverttheprovidedspincolumn(ComponentD)severaltimestore-sUSPendthesettledgelandremoveanybubbles.

3.2Snapoffthetipandplacethecolumninawashingtube(2mL,notprovided).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).

3.3Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.

3.4Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor2minutestoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.

3.5Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.

4.1Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,notprovided).Carefullyloadthesample(~105µL,fromStep2.2)directlytothecenterofthecolumn.

4.2AfterloADIngthesample,add5µLof1XPBS(pH7.2-7.4)tomakethetotalvolumeof110µL.Centrifugethecolumnfor5-6minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredprotein-Buccutite™MTAsolution.

5.1MixwholevialofreconstitutedBuccutite™FOL-ActivatedAPC(ComponentA)withthepurifiedAb-Buccutite™MTAsolution(fromStep4.2),androtatethemixturefor1houratroomtemperature.

5.2TheAb-APCconjugateisnowreadytouse.

Note1:Forimmediateuse,theAPC-Abconjugateneedbedilutedwiththebufferofyourchoice.
Note2:Theconcentrationoftheconjugateisabout0.5~0.6mgAb/mLifstartwith100uL1mg/mlantibodysolution.

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