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AAT Bioquest/Buccutite™ Rapid APC-iFluor™ 700 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 10

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¥143560.00
货号:1319
浏览量:127
品牌:AAT Bioquest
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商品描述
Overview
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Ex/Em(nm)651/713
MWN/A
CAS#N/A
SolventDMSO
StorageF/D/L
CategoryProteinBiochemistry
Generalproteins
Related
APC-iFluor™700TandemisanexcellentreplacementforAPC-AlexaFluor®700Tandemsincetheyhavealmostidenticalspectra.Onsomeantibodies,ourAPC-iFluor™700TandemismuchbrighterthanAPC-AlexaFluor®700Tandemwithahigherstainindex.AATBioquestoffersthisBuccutite™rapidlabelingkittofacilitatetheAPC-iFluor™700tandemconjugationstoantibodiesandotherproteinssuchasstreptavidinandothersecondaryreagents.Buccutite™APC-iFluor™700TandemConjugationKitprovidesarobustandconvenientmethodtoconjugateantibodieswithAPC.ThekitincludesapreactivatedAPC-iFluor™700tandemandreactionbuffer.TheconjugatedantibodycanbeusedinWB,ELISAandIHCapplications.Thiskitissufficientfor2labelingreactions,eachupto100ugofantibody.Thebestratioforanynewantibodyreagentmustbedeterminedbyexperimentation.OurkitprovidespreactivatedAPC-iFluor™700tandemtofacilitatetheAPC-iFluor™700tandemconjugationstoantibodiesandotherproteinssuchasstreptavidinandothersecondaryreagents.OurpreactivatedAPC-iFluor™700tandemisreadytoconjugate,givingmuchhigheryieldthantheconventionallytediousSMCC-basedconjugationchemistry.Inaddition,ourpreactivatedAPC-iFluor™700tandemisconjugatedtoaproteinviaitsaminogroupthatisabundantinproteinswhileSMCCchemistrytargetsthethiolgroupthathastoberegeneratedbythereductionofantibodies.
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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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美国AAT Bioquest Inc.(前身是ABD Bioquest,Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域,包括吸收(颜色),荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学,免疫学,细胞生物学和分子生物学等领域。AAT Bioquest会不断介绍新产品,快速的丰富各个领域的产品。作为AAT Bioquest Inc.的中国区域代理,蚂蚁淘科技为中国客户提供光谱学检测领域,包括底物显色、荧光和发光技术等全系列解决方案。       1、更具品质保障的AAT Bioquest产品(AAT Bioquest作为全球专业的光谱学检测产品生产商,能提供最具性价比的产品); 2、更高价格优势和性价比的选择,我们将一如既往的为客户和代理商提供最具价格竞争力的产品和全面的售后保障(作为全国总代理,我们能提供最大的价格优惠和更全面的技术支持) 3、更快捷的到货时间,我们将以更短的订货周期,更快捷的国际物流以及报关时间为您提供最节省时间的供货保证(蚂蚁淘具有独立的进出口和报关权,因此提供更快捷的到货保障);