| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 565/700 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related |
| Spectrum | AdvancedSpectrumViewer |
- Prepareantibodysolution:
Forlabeling100μgantibody(assumingthetargetantibodyconcentrationis1mg/mL),mix5μL(5%ofthetotalreactionvolume)ofReactionBuffer(ComponentC)with100μLofthetargetantibodysolution.
Note1.Ifyouhaveadifferentantibodyconcentration,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. - RunAntibody-Buccutite™MTAreaction:
- AddtheantibodysolutiondirectlyintothevialofBuccutite™MTA(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
- KeeptheAntibody-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.
Note:TheAntibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.
- PreparespincolumnforAntibody-Buccutite™MTApurification:
- Inverttheprovidedspincolumn(ComponentD)severaltimestore-sUSPendthesettledgelandremoveanybubbles.
- Snapoffthetipandplacethecolumninawashingtube(2mL,notprovided).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
- Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
- Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor2minutestoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
- Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
- PurifytheAb-Buccutite™MTAsolution:
- Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,notprovided).Carefullyloadthesample(~105μL,fromStep2.2)directlytothecenterofthecolumn.
- AfterloADIngthesample,add5μLof1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5-6minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredprotein-Buccutite™MTAsolution.
- MakeAb-PEorPETandemconjugation:
- MixwholevialofBuccutite™FOL-ActivatedPEorPETandem(ComponentA)withthepurifiedAb-Buccutite™MTAsolution(fromStep4.2),androtatethemixturefor1houratroomtemperature.
- TheAb-PEorPETandemconjugateisnowreadytouse.
Note1:Forimmediateuse,theAb-PEorPETandemconjugateneedbedilutedwiththebufferofyourchoice.
Note2:Theconcentrationoftheconjugateisabout0.5~0.6mgAb/mLifstartwith100uL1mg/mlantibodysolution.
| References&Citations |  PrinterFriendlyVersion |
1. ZhaoKH,SuP,LiJ,TuJM,ZhouM,BubenzerC,ScheerH.(2006)Chromophoreattachmenttophycobiliproteinbeta-subunits:phycocyanobilin:cysteine-beta84phycobiliproteinlyaseactivityofCpeS-likeproteinfromAnabaenaSp.PCC7120.JBiolChem,281,8573.
2. PetrasekZ,SchmittFJ,TheissC,HuyerJ,ChenM,LarkumA,EichlerHJ,KemnitzK,EckertHJ.(2005)ExcitationenergytransferfromphycobiliproteintochlorophylldinintactcellsofAcaryochlorismarinastudiedbytime-andwavelength-resolvedfluorescencespectroscopy.PhotochemPhotobiolSci,4,1016.
3. LoosD,CotletM,DeSchryverF,HabuchiS,HofkensJ.(2004)Single-moleculespectroscopyselectivelyprobesdonorandacceptorchromophoresinthephycobiliproteinallophycocyanin.BiophysJ,87,2598.
4. PrasannaR,PrasannaBM,MohammadiSA,SinghPK.(2003)EvaluationofTolypothrixgermplasmforphycobiliproteincontent.FoliaMicrobiol(Praha),48,59.
5. PrasannaR,DharDW,DominicTK,TiwariON,SinghPK.(2003)IsolationandcharacterisationofphycobiliproteinrichmutantofcyanobacteriumSynechocystissp.ActaBiolHung,54,113.
6. WuP.(2000)[Phycobiliproteinandfluorescenceimmunologicalassay].ShengLiKeXueJinZhan,31,82.
7. NoubirS,LuqueI,OchoadeAldaJA,PerewoskaI,TandeaudeMarsacN,CobleyJG,HoumardJ.(2002)Co-ordinatedexpressionofphycobiliproteinoperonsinthechromaticallyadaptingcyanobacteriumCalothrixPCC7601:aroleforRcaDandRcaG.MolMicrobiol,43,749.
8. TingCS,RocapG,KingJ,ChisholmSW.(2001)PhycobiliproteingenesofthemarinephotosyntheticprokaryoteProchlorococcus:evidenceforrapidevolutionofgeneticheterogeneity.MicroBIOLOGy,147,3171.
9. TriantafilouK,TriantafilouM,WilsonKM.(2000)Phycobiliprotein-Fabconjugatesasprobesforsingleparticlefluorescenceimaging.Cytometry,41,226.
10. ZhaoKH,DengMG,ZhengM,ZhouM,ParbelA,StorfM,MeyerM,StrohmannB,ScheerH.(2000)Novelactivityofaphycobiliproteinlyase:boththeattachmentofphycocyanobilinandtheisomerizationtophycoviolobilinarecatalyzedbytheproteinsPecEandPecFencodedbythephycoerythrocyaninoperon.FEBSLett,469,9.
