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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
产品编号:36381
市  场 价:¥70000.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$3500.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Screen Quest™ FRET No Wash cAMP Assay Kit/36381/50 plates
商品介绍
Overview
PrinterFriendlyVersion

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)


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