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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
商品详细AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
商品编号: 36381
品牌: aatbio
市场价: ¥70000.00
美元价: 42000.00
产地: 美国(厂家直采)
公司:
产品分类: 其它检测试剂盒
公司分类: Other_kits
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍
Overview
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Ex/Em(nm)390/650
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryGPCR
cAMPGPCRAssays
RelatedDiagnosticMolecules
CellSignaling
BiochemicalAssays
ScreenQuest™NoWashFRETcAMPAssayKitprovidesaconvenientassaymethodformonitoringtheactivationofadenylylcyclaseinG-proteincoupledreceptorsystems.ComparedtoothercommercialELISAcAMPassaykits,thishomogenouscAMPassaykitdoesnotrequireawashsteportheacetylationstep.Theassayisbasedonthecompetitionforafixednumberofanti-cAMPantibodybindingsitesbetweenthefluorescentcAMPtracerandnon-labeledfreecAMP.FreecAMPdisplacesthefluorescentcAMPtracerfromtheHRP-cAMP/anti-cAMPantibodycomplex.Theanti-cAMPantibodyislabeledwithourTRFluor™EuwhilethecAMPtracercontainsourbFluor™650.IntheabsenceofcAMP,bFluor™650-cAMPconjugateisboundtoTRFluor™Eu-labeledanti-cAMPantibodyexclusivelytohaveastrongFRET.However,theunlabeledfreecAMPinthetestsamplecompetesfortheTRFluor™Eu-labeledanti-cAMPantibodyconjugate,thereforeinhibitsthebindingofbFluor™650-cAMPtoanti-cAMPantibody.ThebFluor™650labeledcAMPtraceronlyhasfluorescencelifetimeofnanosecondwhileTRFluor™Eu-labeledanti-cAMPantibody-boundfluorescentcAMPtracerhasmuchlongerfluorescencelifetimevalueduetotheTR-FRET.ThemagnitudeofFRETisproportionaltotheconcentrationofcAMPinasample.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformat.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
1.Preparesamples:

Foradherentcells:Platecellsovernightingrowthmediumat30,000-100,000cells/wellfora96-wellplate.

Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat100,000-300,000cells/wellfora96-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiment.

Treatcellsasdesired:ThefollowingisanexampleforHelacellstreatedwithForskolintoinducecAMPina96-wellplateformat.25µLcellsingrowthmedium,add25µL/well100µMForskolininHanksand20mMHepesbuffer(HHBS),incubateina5%CO2,37°Cincubatorfor15minutes.

Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.

2.PreparecAMPStandard:

Add100µLComponentE(Diluent)toComponentC(cAMPStandard)tomake1mMstocksolution.Dilutioncanbecarriedoutwiththediluent(orwithcellculturemedia).Add2.24µL1mMstocksolutioninto200µLDiluenttomake11200nMstandard,andthenget50µL11200nMcAMPstandardinto150µLDilenttoget2800nMStandard,andrepeatthisproceduretomaketheserialdilutions.ThecAMPstandarddilutionsare:11200,2800,700,175,43.75,10.94,2.73,0.68nM(finalconcentrationof2800~0.17nMcAMPperwell).

3.PrepareAnticAMP-trFluor™Euworkingsolution:

Add55µLComponentEintovialAtoreconstituteAnticAMP-trFluor™Eufor#36379,(or550µLComponentEintovialAfor#36380and#36381),andthenprepareworkingsolutionbyadding50µLreconstitutedComponentAto2.5mLCellLysisBuffer(ComponentD),ormaketheneededvolumeproportionally.Preparebeforeuse!

4.PreparecAMP-trFluor™650workingsolution:

Add55µLComponentEintovialBtoreconstitutecAMP-trFluor™650for#36379,(or550µLComponentEintovialAfor#36380and#36381),andthenprepareworkingsolutionbyadding50µLreconstitutedComponentBto2.5mLCellLysisBuffer(ComponentD),ormaketheneededvolumeproportionally.Preparebeforeuse!

5.RuncAMPassay:

RuncAMPAssay(includingthestandardcurveandcellbasedassay)asinstructioninthefollowingtable:

cAMPStandardCells
NegativeControlPositiveControlStandardCurveNegativeControlNon-stimulatedStimulated
25µLDiluent25µLDiluent25µLStandard25µLCells25µLCells25µLCells
25µLCompoundBuffer25µLCompoundBuffer25µLCompoundBuffer25µLCompoundBuffer25µLCompoundBuffer25µLCompound
Incubate30minatRT
25µLLysisBuffer25µLcAMP-trFluor™650workingsolution25µLcAMP-trFluor™650workingsolution25µLLysisBuffer25µLcAMP-trFluor™650workingsolution25µLcAMP-trFluor™650workingsolution
25µLAnticAMP-trFluor™Euworkingsolution
Incubate30minatRT

ReadonacompatIBLeTR-FRETreader.
References&Citations
CitationExplorer

ActivationofP2X7andP2Y11purinergicreceptorsinhibitsmigrationandnormalizestumor-derivedendothelialcellsviacAMPsignaling
Authors:DAvanzato,TGenova,AFiorioPla,MBernardini,SBianco,BBussolati,DMancardi,EGiraudo,FMaione,PCassoni
Journal:ScientificReports(2016)

TheM2muscarinicreceptorsareessentialforsignalingintheheartleftventricleduringrestraintstressinmice
Authors:HanaTomankova,PaulinaValuskova,EvaVarejkova,JanaRotkova,JanBenes,JaromirMyslivecek
Journal:Stress(2015)


品牌介绍
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会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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