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AAT Bioquest/Buccutite™ Rapid Protein Crosslinking Kit *Microscale Optimized for Crosslinking 100 ug Antibody Per Reac
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R |
| Category | ProteinBiochemistry Generalproteins |
| Related |
Uponreceipt,storecomponentAandBat-20oC.Whenstoredproperly,thekitshouldbestableforsixmonths.DonotfreezeReactionBuffer(ComponentC)andSpinColumn(ComponentD).Warmallthecomponentsandcentrifugethevialsbrieflybeforeopening,andimmediatelypreparetherequiredsolutionsbeforestartingyourconjugation.ThefollowingSOPisanexampleforconjugate100μgprotein-1withprotein2.
1.PrepareProteinSolution:Forlabeling100μgprotein-1andprotein-2(assumingtheconcentrationsare1mg/mLforbothproteins),mix5μL(5%ofthetotalreactionvolume)ofReactionBuffer(ComponentC)with100μLoftheeachproteinsolution.Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Ifitisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.StABIlizerslikebovineserumalbumin(BSA)orgelatinwillaffectthelabelingreaction.
2.RunProtein1-Buccutite™MTAReaction:
2.1Add105μLProtein-1solutiondirectlyintothevialofBuccutite™MTA(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
2.2KeeptheProtein-1-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.TheAntibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.
2.3PurifyProtein-1-Buccutite™MTAthroughdesaltingcolumn.Pleasereferto“PreparespincolumnforAntibodypurification”partfordetailedoperations.
2.4CalculatetheconcentrationoftheProtein-1-Buccutite™MTAwith75%yieldafterdesalting.(Forexample:ifstartingwith100μgprotein,afterdesaltingcolumnpurification,therecoveryproteinamountis~75μg.)
3.RunProtein2-Buccutite™FOLreaction:
3.1Addthe105μLProtein-2solutiondirectlyintothevialofBuccutite™FOL(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
3.2KeeptheProtein-2-Buccutite™FOLreactionmixtureatroomtemperaturefor30-60minutes.TheAntibody-Buccutite™FOLreactionmixturecanberotatedorshakenforlongertimeifdesired.
3.3PurifyProtein-2-Buccutite™FOLthroughdesaltingcolumn.Pleasereferto“PreparespincolumnforAntibodypurification”partfordetailedoperations.
3.4CalculatetheconcentrationoftheProtein-2-Buccutite™FOLwith75%yieldafterdesalting.
4.Cross-linkingProtein-1andProtein-2Reaction:
4.1Cross-linkingreactionisinitiatedbymixingProtein-1-Buccutite™MTAandProtein-2-Buccutite™FOLatthedesiredmolarratio.Usually,Buccutite™FOL-modifiedProtein-2ismixedwithBuccutite™MTA-modifiedProtein-1in1.5-2.0molarratiotodrivethecrosslinkingreactiontocompletion.Themixingratiocanbereverseddependingonyourdownstreamapplications,andthecostofyourproteins.
4.2Rotatethemixturefor1~2houratroomtemperatureorstayat4oCovernight.Thereactionmixtureisnowreadytouse,ortobestoredat4oC.Desaltingisoptional.
5.Optional:ConjugateAnalysisandPurification:
5.1Asmallamountofreactionmixture(forexample:2~4μg)couldbeanalyzedusing4-12%Bis-TisProteinGelinaSDSrunningbuffersystemtochecktheconjugationresult.
5.2TheconjugationreactionmixturecontainsthedesiredconjugatealongwithsmallamountofunlinkedunlinkedProtein-2(usedinexcess).Ifrequired,thereactionmixturecanbepurifiedbysizeexclusionchromatography(SEC),andthedesiredconjugatefractionsarepooledandcombined.
PreparespincolumnforAntibodypurification:
1.Inverttheprovidedspincolumn(ComponentD)severaltimestore-sUSPendthesettledgelandremoveanybubbles.
2.Snapoffthetipandplacethecolumninawashingtube(2mL,notprovided).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
3.Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor2minutestoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
5.Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
6.Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,notprovided).Carefullyloadthesample(~105μL,fromStep2.2)directlytothecenterofthecolumn.
7.AfterloADIngthesample,add10μLof1XPBS(pH7.2-7.4),centrifugethecolumnfor5-6minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredprotein-1-Buccutite™MTAsolutionorProtein-2-Buccutite™FOLsolution.
| References&Citations |  PrinterFriendlyVersion |
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7. Holloway,J.L.;Ma,H.;Rai,R.;Burdick,J.A.,Modulatinghydrogelcrosslinkdensityanddegradationtocontrolbonemorphogeneticproteindeliveryandinvivoboneformation.JControlRelease2014,191,63-70.
8. Luo,C.;Zhao,J.;Tu,M.;Zeng,R.;Rong,J.,Hyaluronanmicrogelasapotentialcarrierforproteinsustaineddeliverybytailoringthecrosslinknetwork.MaterSciEngCMaterBiolAppl2014,36,301-8.
9. Lee,H.;Alpi,A.F.;Park,M.S.;Rose,A.;Koo,H.S.,C.elegansringfingerproteinRNF-113isinvolvedininterstrandDNAcrosslinkrepairandinteractswithaRAD51Chomolog.PLoSOne2013,8(3),e60071.
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