ProductName | CyclicAMPCLIAKit(High-Sensitivity) |
Description | ChemiluminescentdetectionofcAMP |
SpeciesReactivity | SpeciesIndependent |
Platform | Microplate |
SampleTypes | Celllysates,EDTAPlasma,HeparinPlasma,Saliva,Tissue,TissueCultureMedia,Urine |
DetectionMethod | ChemiluminescentAssay |
AssayType | DirectCLIA(ChemiluminescentImmunoassay) |
Utility | CLIAkitusedtoquantitativelymeasurecAMPpresentinsamples. |
Sensitivity | 0.119pmol/ml |
AssayRange | Regular:0.185-15pmol/ml.Acetylated:0.039-5pmol/ml |
Precision | IntraAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-11.2pmol/mL,3.9%CVSample2-3.6pmol/mL,8.3%CVIntraAssayPrecision(Acetylated):TwohumanplasmasamplesweredilutedwithSampleDiluent,acetylatedandruninreplicatesof20inanassay.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-1.32pmol/mL,8.4%CVSample2-0.39[pmol10.9%CVInterAssayPrecision(Regular):TwohumanurinesamplesweredilutedwithSampleDiluentandruninduplicatesinfourteenassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-6.5pmol/mL,8.1%CVSample2-2.1pmol/mL,13.0%CVInterAssayPrecision(Acetylated):TwohumanplasmasampleweredilutedwithSampleDiluent,acetylatedandruninduplicatesinfourteenassaysrunovermultipledaysbyfouroperators.ThemeanandprecisionofthecalculatedcAMPconcentrationswere:Sample1-1.02pmol/mL,10.1%CVSample2-0.32pmol/mL,16.5%CV |
IncubationTime | 2hours |
NumberofSamples | 38samplesinduplicate |
OtherResources | KitBooklet,MSDS,SalivaSampleHandlingInstructions |
StorageTemperature | 4ºC | ||||||||||||||||||||||||||||||||||||||||||
ShippingTemperature | BlueIce | ||||||||||||||||||||||||||||||||||||||||||
ProductType | CLIAKits | ||||||||||||||||||||||||||||||||||||||||||
AssayOverview | TheCyclicAMP(cAMP)CLIA(High-Sensitivity)kitisdesignedtoquantitativelymeasurecAMPpresentinlysedcells,EDTAandheparinplasma,urine,salivaandtissueculturemediasamples.Fortissuesamples,salivaandurine,wherethelevelsofcAMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.Forplasmasamplesandsomedilutecelllysatesanoptionalacetylationprotocolcanbeused.Thiskitcanmeasureaslittleas1femtomolcAMPpersample.ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstABIlizers,forcAMPmeasurement.Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.AcidifiedsamplesofcAMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.AcAMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.AwhitemicrotiterplatecoatedwithanantibodytocapturesheepIgGisprovided.PriortotheadditionofanysamplesorstandardsaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.Standardsordilutedsamples,eitherwithorwithoutacetylation,arePipettedintotheprimedwells.AcAMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.ThebindingreactionisinitiatedbytheadditionofasheepantibodytocAMPtoeachwell.Aftera2hourincubation,theplateiswashedandthechemiluminescentsubstrateisadded.ThesubstratereactswiththeboundcAMP-peroxidaseconjugatetoproducelightThegeneratedlightisdetectedinamicrotiterplatereadercapableofreADIngluminescence.TheconcentrationofthecAMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders. | ||||||||||||||||||||||||||||||||||||||||||
KitOverview |
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CiteThisProduct | CyclicAMPCLIAKit(High-Sensitivity)(StressMarqBiosciencesInc.,VictoriaBCCANADA,Catalog#SKT-208) |
AlternativeNames | 3"-5"-cyclicadenosinemonophosphateCLIAKit |
ResearchAreas | CellSignaling,Neuroscience |
ScientificBackground | Adenosine-3’,5’-cyclicmonophosphate(cyclicAMP)isasecondmessengerthatplaysanimportantroleinintracellularregulation.Specifically,itfunctionsasamediatorofactivityofseveralkeyhormonesincludingepinephrine,glucagon,andACTH(1-4).AdenylatecyclaseisactivatedbythehormonesglucagonandadrenalineandbyGprotein.Liveradenylatecyclaserespondsmorestronglytoglucagon,andmuscleadenylatecyclaserespondsmorestronglytoadrenaline.cAMPdecompositionintoAMPiscatalyzedbytheenzymephosphodiesterase.IntheHumanMetabolomeDatabasethereare166metabolicenzymeslistedthatconvertcAMP(5).OtherbiologicalactionsofcAMPincluderegulationofinnateimmunefunctioning(6),axonregeneration(7),cancer(8),andinflammation(9). |
References | 1.Sutherland,E.W.andRall,T.W.FractionationandCharacterizationofaCyclicAdenineRibonucleotideFormedbyTissueParticles.J.Biol.Chem.,232:1077,1958. 2.Marsh,J.M.Biol.Reprod.,14:30-53,1976. 3.Korenman,S.G.andKrall,J.F.Biol.Reprod.,16:1-17,1977. 4.Kelley,D.J.,Bhattacharyya,A.,Lahvis,G.P.,Yin,J.C.P.,Malter,J.,andDavidson,R.J.Neurosci.Biobehav.Rev.,32(8):1533-1543,2008. 5.http://www.hmdb.ca/metabolites/HMDB00058 6.Serezani,C.H.,Ballinger,M.N.,Aronoff,D.M.,andPeters-Golden,M.Am.J.Resp.CellandMol.Biol.,39(2):127,2008. 7.Hannila,S.S.,andFilbin,M.T.Exp.Neurol.,209(2):321–332,2008. 8.Shankar,D.B,Cheng,J.C.,andSakamoto,K.M.Cancer,104(9):1819-24,2005. 9.GaleaE.andFeinstein,D.L.FASEBJ.,13:2125-2137,1999. |
RegularFormatTypicalStandardCurvefortheCyclicAMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-208.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.185–15pmol/ml.Acetylated:0.039–5pmol/ml.
AcetylatedFormatTypicalStandardCurvefortheCyclicAMPCLIAKit(High-Sensitivity)(ChemiluminescentImmunoassay)StressXpress®–SKT-208.AssayType:DirectCLIA.DetectionMethod:ChemiluminescentAssay.AssayRange:Regular:0.185–15pmol/ml.Acetylated:0.039–5pmol/ml.
LinearitywasdeterminedbytakingtwoJurkatcelllysatesamples,onewithalowcAMPlevelof0.2pmol/mLandonewithahigherlevelof2.5pmol/mL,andmixingthemintheratios.Themeasuredconcentrationswerecomparedtotheexpectedvaluesbasedontheratiosused.
ChemicalstructureofCyclicAMP(cAMP)
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