| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R/D/L |
| Category | GPCR cAMPGPCRAssays |
| Related | DiagnosticMolecules CellSignaling BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
Note1.Allowallthekitcomponentstowarmtoroomtemperaturebeforeusingthem;Note2:Somematerialmightbesticktothevialcapduringtheshipment.Brieflycentrifugethevialtocollectallthecontent.
1.Preparesamples:
1.1 CellSamples:
Foradherentcells:Platecellsovernightingrowthmediumat30,000-100,000cells/wellfora96-wellplate.
Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat100,000-300,000cells/wellfora96-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Treatcellsasdesired:ThefollowingisanexampleforHelacellstreatedwithFoskolintoinducecAMPina96-wellplateformat.
a).Aspirateoffcellgrowthmedium,add100µL/well100µMForskolininHanksand20mMHepesbuffer(HHBS),incubateina5%CO2,37oCincubatorfor15minutes;b).Aspirateoffcellsolutionaftertheincubation,add100µL/wellofCellLysisBuffer(ComponentE),andincubateatroomtemperatureforanother10minutes.ThiscelllysatecanbeassayeddirectlyordilutedinAssayBuffer(ComponentB).
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
1.2 TissueSamples:Itisimportanttorapidlyfreezetissuesaftercollection(e.g.,usingliquidnitrogen)duetoquickmetabolismofcyclicnucleotidesintissue.Weighthefrozentissueandadd10-20μL/mgofcelllysisbuffer.Homogenizethesampleonice.Spinattopspeedfor5minutesandcollectthesupernatant.Thesupernatantmaybeassayeddirectly.
1.3 Urine,PlasmaandCultureMediumSamples:Urineandplasmamaybetesteddirectlywith1:200to1:1000dilutionsin1XLysisBuffer.Culturemediumcanalsobetestedwith1:10to1:200dilutionsinLysisBuffer.
Note:RPMImediummaycontain>350fmol/μLcAMP.
2.PreparecAMPassaysolutions:
2.1 Prepare100µMcAMPstocksolutionbyadding1mLofAssayBuffer(ComponentB)tothevialofcAMPStandard(ComponentA).Make1:10,1:100and1:3serialdilutionsinAssayBuffer(ComponentB)tohave10,000,100,30,10,3,1,0.3,and0nMcAMPdilutedsolutions.Storeoniceor4oC.
Note:Theunusedreconstituted100µMcAMPstocksolutionshouldbealiquotedandstoredat-20oC.
2.2 Prepare50XHRP-cAMPconjugatestocksolutionbyadding55µL(forKit36373)or550µL(forKit36374)ofAssayBuffer(ComponentB)intothevialofHRP-cAMPConjugate(ComponentC).Make1:50dilutionwithassaybuffertohave1XHRP-cAMPconjugateworkingsolutionbeforeuse.Storeitoniceor4oC.
Note1:25µLof1XHRP-cAMPconjugateworkingsolutionisenoughforoneassaypoint;prepareappropriatelyvolumeforsingleuseonly;
Note2:Theunused50XHRP-cAMPconjugatestocksolutionshouldbedividedintosingleusealiquotsandstoredthemat-20oC.
2.3 Prepare1Xwashingsolutionbyadding1mLof10XWashSolution(ComponentD)to9mLdistilledwater.
2.4 Prepare200XAmplite™Redstocksolutionbyadding50µL(forKit36373)or500µL(forKit36374)ofDMSOintothewellofAmplite™Red(ComponentG).
Note:0.5µLof200XAmplite™Redstocksolutionisenoughforoneassaypoint.Theunusedreconstitutedstocksolutionshouldbealiquotedandstoredat-20oC.
3.RuncAMPassay:
3.1 Alltheassaywellswillbepreparedinthefollowingorders:A)cAMPstandards,control,ortestssamples;B)HRP-cAMPconjugate.
3.2 Add75µL/wellofthecAMPdilutedsolution(fromStep2.1)andtestsamplesintoeachwelloftheanti-cAMPAbcoated96-wellplate(ComponentH).Itisrecommendedtoduplicatetheassaysforeachstandardandtestsample.Incubateatroomtemperaturefor5to10minutes.
3.3 Add25µL/wellof1XHRP-cAMPconjugateworkingsolution(fromStep2.2).Incubateatroomtemperaturefor2hoursbyplacingtheplateonshaker.
3.4 Aspirateplatecontents,andwash4timeswith200µL/wellof1Xwashsolution(fromStep2.3).
3.5 PrepareAmplite™Redworkingsolutionbyadding50µLof200XAmplite™Redstocksolution(fromStep2.4)and11.5µLof3%H2O2(ComponentF)into10mLofSubstrateBuffer(ComponentI).
Note:TheAmplite™Redworkingsolutionisnotstable,useitpromptly.
3.6 Add100µL/wellofAmplite™Redworkingsolution(fromStep3.5)intoeachwell,andincubateatroomtemperaturefor10minutesto2hours,orupto24hours,protectedfromlight.
3.7 MonitorthefluorescenceincreaseatEx/Em=540/590nm(cutoff570nm)byusingafluorescenceplatereader(topreadmode).
| 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 |
本网站将在规定时间内给予删除等相关处理。
