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蚂蚁淘在线 / 品牌中心 / AAT Bioquest / AAT Bioquest/Buccutite&trade；快速APC-Cy7串联抗体标记试剂盒*用于标记100μg抗体的微型优化

AAT Bioquest/Buccutite&trade；快速APC-Cy7串联抗体标记试剂盒*用于标记100μg抗体的微型优化

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￥143560.00
货号：1321
浏览量：127
品牌：AAT Bioquest
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Ex/Em(nm)651/780
MWN/A
CAS#N/A
SolventDMSO
StorageF/D/L
CategoryProteinBiochemistry
Generalproteins
Related
APC-Cy7isapopularcolorusedinflowcytometry.Itsprimaryabsorptionpeakisat651nmwithemissionpeakat~780nm.AATBioquestoffersthisBuccutite™rapidlabelingkittofacilitatetheAPC-Cy7tandemconjugationstoantibodiesandotherproteinssuchasstreptavidinandothersecondaryreagents.Buccutite™APC-Cy7TandemConjugationKitprovidesarobustandconvenientmethodtoconjugateantibodieswithAPC.ThekitincludesapreactivatedAPC-Cy7tandemandreactionbuffer.TheconjugatedantibodycanbeusedinWB,ELISAandIHCapplications.Thiskitissufficientfor2labelingreactions,eachupto100ugofantibody.Thebestratioforanynewantibodyreagentmustbedeterminedbyexperimentation.OurkitprovidespreactivatedAPC-Cy7tandemtofacilitatetheAPC-Cy7tandemconjugationstoantibodiesandotherproteinssuchasstreptavidinandothersecondaryreagents.OurpreactivatedAPC-Cy7tandemisreadytoconjugate,givingmuchhigheryieldthantheconventionallytediousSMCC-basedconjugationchemistry.Inaddition,ourpreactivatedAPC-Cy7tandemisconjugatedtoaproteinviaitsaminogroupthatisabundantinproteinswhileSMCCchemistrytargetsthethiolgroupthathastoberegeneratedbythereductionofantibodies.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
1.Prepareantibodysolution:

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.RunAntibody-Buccutite™MTAreaction:

2.1AddtheantibodysolutiondirectlyintothevialofBuccutite™MTA(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.

2.2KeeptheAntibody-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.

Note:TheAntibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.

3.PreparespincolumnforAntibody-Buccutite™MTApurification:

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.PurifytheAb-Buccutite™MTAsolution:

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.MakeAb-APCconjugation:



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
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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.


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AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。AAT Bioquest拥有超过5000项产品，包括Fluo-8®、Cal-520®、Cell Meter™、Amplite™、CytoTrace™和CytoTell™等产品线，这些品牌在生命科学研究领域获得了认可和好评。 AAT bioquest 提供酶、核酸、蛋白标记、小分子检测、细胞凋亡、钙离子、离子通道、膜电位检测等特色试剂盒及荧光探针。AAT Bioquest提供的客户定制服务包括比色、荧光和发光探针的合成，生化、细胞和诊断检测试剂的开发，以及使用AAT Bioquest已经验证的化合物库来筛选客户的目标化合物等。   l   酶的活性检测 l   核糖核酸检测 l   蛋白质化学 l   小分子检测 l   荧光标记探针 l   生物素及其衍生物 l   经典荧光标记染料 l   偶联剂 l   荧光淬灭剂 l   特优荧光标记染料   l   蛋白标记