| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 651/713 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related |
| Spectrum | AdvancedSpectrumViewer |
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.
| References&Citations |  PrinterFriendlyVersion |
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