Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 390/650 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | GPCR cAMPGPCRAssays |
Related | DiagnosticMolecules CellSignaling BiochemicalAssays |
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.
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:
cAMPStandard | Cells | ||||
NegativeControl | PositiveControl | StandardCurve | NegativeControl | Non-stimulated | Stimulated |
25µLDiluent | 25µLDiluent | 25µLStandard | 25µLCells | 25µLCells | 25µLCells |
25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompound |
Incubate30minatRT | |||||
25µLLysisBuffer | 25µLcAMP-trFluor™650workingsolution | 25µLcAMP-trFluor™650workingsolution | 25µLLysisBuffer | 25µLcAMP-trFluor™650workingsolution | 25µLcAMP-trFluor™650workingsolution |
25µLAnticAMP-trFluor™Euworkingsolution | |||||
Incubate30minatRT |
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)
AAT Bioquest常用产品报价单
产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |