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AntibodiesOnline/cAMP ELISA Kit (Cyclic Adenosine Monophosphate)/ABIN577668/96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/cAMP ELISA Kit (Cyclic Adenosine Monophosphate)/ABIN577668/96 tests
Antigen
CyclicAdenosineMonophosphate(cAMP)
  • CRAMP
  • CAP-18
  • CAP18
  • FALL-39
  • FALL39
  • LL37
  • CAP11
  • Cnlp
  • Cramp
  • MCLP
  • CATHL7
  • cathelicidinantimicrobialpeptide
  • Camp
  • CAMP
Alternatives
ChemicalcAMPELISAKit
Kitswithalternativereactivityto:
28Chemical
3Mouse(Murine)
2Human
1Chicken
1Cow(Bovine)
1DNA
1Dog(Canine)
1General
1Goat
1GuineaPig
1Monkey
1Pig(Porcine)
1Rabbit
1Rat(Rattus)
1Sheep(Ovine)
MethodType
SandwichELISA
MinimumDetectionLimit
< 1 fM cAMP/Sample
Application
ELISA
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®DirectHighSensitivityCyclicAMP(cAMP)ChemiluminescentImmunoassaykitisdesignedtoquantitativelymeasurecAMPpresentinlysedcells,EDTAandheparinplasma,urine,salivaandtissueculturemediasamples.
BrandDetectX®
SampleTypeCellLysate,Saliva,Urine,Plasma(EDTA),Plasma(heparin),TissueCultureMedium
AnalyticalMethodQuantitative
DetectionMethodChemiluminescent
SpecificitySpeciesIndependent.SamplesTypesvalidated:CellLysates,Saliva,Urine,EDTAandHeparinPlasma,TissueCultureMedia
Cross-Reactivity(Details)(%)CyclicAMP100 %AMP<0.08 %GMP<0.08 %CyclicGMP<0.08 %ATP<0.08 %
Sensitivity0.119pmol/mL
CharacteristicsTheCyclicAMP(cAMP)ChemiluminescentDirectImmunoassay(CLIA)kitsaredesignedtoquantitativelymeasurecAMPpresentincelllysates,plasma,urine,saliva,tissueandculturemediasamples.ThesuppliedSampleDiluentwilllysecells,stabilizecAMPandstopphosphodiesteraseactivity.AcAMPstandardisprovidedtogenerateastandardcurvefortheassay.ThesuppliedPlatePrimersolutionisaddedtothewellsofacoatedwhitemicrotiterplate,followedbystandardsordilutedsamples.AcAMP-peroxidaseconjugateisthenaddedtothewells.ThebindingreactionisinitiatedbytheadditionofapolyclonalantibodytocAMP.Aftera2hourincubationtheplateiswashedandchemiluminescentsubstrateisadded.ThesubstrateimmediatelyreactswiththeboundcAMP-peroxidaseconjugate.Thegeneratedchemiluminescentglowsignalismeasured.TheconcentrationofthecAMPinthesampleiscalculated,aftermakingcorrectionforthedilution.CyclicAMP(cAMP)isoneofthemostimportantsecondmessengersandakeyintracellularregulator.DiscoveredbySutherlandandRallin1957,itfunctionsasamediatorofactivityforanumberofhormones,includingepinephrine,glucagon,andACTH.CyclicAMPisproducedbytheenzymeadenylatecyclase,andtheenzymeisactivatedbythehormonesglucagonandadrenalineandbyGprotein.cAMPdecompositionintoAMPiscatalyzedbytheenzymephosphodiesterase.OtherbiologicalactionsofcAMPincluderegulationofinnateimmunefunctioning,axonregeneration,cancer,andinflammation.
ComponentsCoatedWhite96WellPlatesWhiteplasticmicrotiterplate(s)coatedwithdonkeyanti-sheepIgG.1Or5Each
CyclicAMPStandard125μLCyclicAMPat1,500pmol/mLinaspecialstabilizingsolution.
DetectX®CyclicAMPAntibodyAsheepantibodyspecificforcyclicAMP.3mLOr13mL
DetectX®CyclicAMPConjugateConcentrateA50XcyclicAMP-peroxidaseconjugateconcentratestockinaspecialstabilizingsolution.60μLOr260μL
ConjugateDiluentContainsspecialstabilizersandadditives.3mLOr13mL
SampleDiluentConcentrateNowSuppliedONLYasConcentrateContainsspecialstabilizersandadditives.The4Xconcentratemustbedilutedwithdeionizedordistilledwater.CAUSTIC12mLOr60mL
PlatePrimerAneutralizingsolutioncontainingspecialstabilizersandadditivies.25mLAceticAnhydride2mLAceticAnhydrideWARNING:CorrosiveLachrymatorTriethylamine4mLTriethylamineWARNING:CorrosiveLachrymator
WashBufferConcentrateA20Xconcentratethatmustbedilutedwithdeionizedordistilledwater.30mLOr125mL
SubstrateSolutionA6mLOr28mL
SubstrateSolutionB6mLOr28mL
PlateSealer1Or5Each
MaterialnotincludedDistilledordeionizedwater.
Repeaterpipetanddisposabletipscapableofdelivering25and100μL.
Microplateshaker.96wellmicroplatereadercapableofreadingglowchemiluminescence.
Alistofsomemodelsofsuitablereaderscanbefoundonourwebsiteatwww.ArborAssays.com/resources/lit.asp.
AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
ThenumberofRLUsobtainedisdependantonthesensitivityandgainofthereaderused.
Ifyouareunsureofhowtoproperlyconfigureyourreadercontactyourplatereadermanufac-turerorcarryoutthefollowingprotocol:Dilute5μLoftheCyclicAMPConjugateConcentrateinto995μLofdeionizedwater.
Pipet5μLofdilutedconjugateinto245μLofdeionizedwater.
Pipet5μLofthismixtureintoawhitewellandadd100μLofpreparedCLIAsubstrate(seepage9fordetails).
Thiswellwillgiveyouanintensityslightlyabovethemaximumbindingfortheassay.
Adjustthegainorsensitivitysothatyourreaderisgivingclosetothereadersmaximumsignal.
ToproperlyanalyzethedatasoftwarewillberequiredforconvertingrawRLUreadingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
AlternativeNameCyclicAMP(cAMPELISAKitAbstract)
TargetTypeDNA
BackgroundAdenosine-3,5-cyclicmonophosphate,orcyclicAMP(cAMP),C10H12N5O6P,isoneofthemostim-portantsecondmessengersandakeyintracellularregulator.DiscoveredbySutherlandandRallin19571,itfunctionsasamediatorofactivityforanumberofhormones,includingepinephrine,glucagon,andACTH2-4.AdenylatecyclaseisactivatedbythehormonesglucagonandadrenalineandbyGprotein.Liveradenylatecyclaserespondsmorestronglytoglucagon,andmuscleadeny-latecyclaserespondsmorestronglytoadrenaline.cAMPdecompositionintoAMPiscatalyzedbytheenzymephosphodiesterase.IntheHumanMetabolomeDatabasethereare166metabolicenzymeslistedthatconvertcAMP5.OtherbiologicalactionsofcAMPincluderegulationofinnateimmunefunctioning6,axonregen-eration7,cancer8,andinflammation9
PathwaysCellularResponsetoMoleculeofBacterialOrigin
ApplicationNotesThisassayhasbeenvalidatedforlysedcells,saliva,urine,EDTAandheparinplasmasamplesandfortissueculturemediasamples.
Samplesshouldbestoredat-70 °Cforlongtermstorage.24-Hoururinesamplesmayneedtohave1 mLconcentratedhydrochloricacidaddedforevery100 mLvolumetoactasapreservative.
Samplescontainingvisibleparticulateshouldbecentrifugedpriortousing.
CyclicAMPisidenticalacrossallspeciesandweexpectthiskitmaymeasurecAMPfromsourcesotherthanhuman.
TheendusershouldevaluaterecoveriesofcAMPinothersamplesbeingtested.
AfterdilutionintheSampleDiluent(seepage9)theremaybesomeprecipitationofproteins.
Thisprecipitatewillnoteffecttheresultsobtained.
AfterbeingdilutedinSampleDiluentthesamplescanbeassayeddirectlywithin2hours,orfrozenat≤-70 °Cforlateranalysis.
Severelyhemolyzedsamplesshouldnotbeusedinthiskit.
ForsamplescontaininglowlevelsofcAMPandforallplasmasamples,theacetylatedassayprotocolmustbeusedduetoitsenhancedsensitivity.
Allstandardsandsamplesshouldbedilutedinglasstesttubes.
Comment

Samplevalues:Fourteenhumanplasmasamplesweretestedintheassay.
DilutedsampleswereacetylatedandrunintheAcetylatedFormat.
Valuesrangedfrom12.5to43.32pmol/mLwithanaver-ageforthesamplesof21.15pmol/mL.
ThenormalreferencerangeforcAMPinplasmais3.9-13.7pmol/mL10.
Fourhumanurinesampleswerediluted>1:30inSampleDiluentandvaluesrangedintheneatsamplesfrom1,099to4,585pmol/mLwithanaverageforthesamplesof3,034pmol/mL.
ThenormalreferencerangeforcAMPinurineis800-12,000pmol/mL11.
Twohumansalivasampleswerediluted1:4inSampleDiluentandruninboththeRegularandAcetylatedFormats.
Valuesrangedfrom5.8to6.6pmol/mLwithanaverageof6.2pmol/mLintheneatsamples.
ThenormalrangeforcAMPinsalivais3.4-17.2pmol/mL12.

AssayTime2h
PlatePre-coated
ProtocolFortissuesamples,salivaandurine,wherethelevelsofcAMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.
Forplasmasamplesandsomedilutecelllysatesanoptionalacetylationprotocolcanbeused.
Thiskitcanmeasureaslittleas1femtomolcAMPpersample.
ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstabilizers,forcAMPmeasurement.
Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.
AcidifiedsamplesofcAMParesta-bleandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.
AcAMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestan-dardcurve.
AwhitemicrotiterplatecoatedwithanantibodytocapturesheepIgGisprovided.
PriortotheadditionofanysamplesorstandardsaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.
Standardsordilutedsamples,eitherwithorwithoutacetylation,arepipettedintotheprimedwells.
AcAMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.
ThebindingreactionisinitiatedbytheadditionofasheepantibodytocAMPtoeachwell.
Aftera2hourincubation,theplateiswashedandthechemiluminescentsubstrateisadded.
ThesubstratereactswiththeboundcAMP-peroxidaseconjugatetoproducelightThegeneratedlightisdetectedinamicrotiterplatereadercapableofreadingluminescence.
TheconcentrationofthecAMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30-60minutes.
WerecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydeterminecAMPconcentrations.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.
WashBufferDiluteWashBufferConcentrate1:20byaddingonepartoftheconcentratetonineteenpartsofdeionizedwater.
Oncedilutedthisisstableatroomtemperaturefor3months.
SampleDiluentNowSuppliedONLYasConcentratePreparetheSampleDiluentbydilutingtheSampleDiluentConcentrate1:4,addingonepartoftheconcentratetothreepartsofdeionizedwater.
Oncedilutedthisisstableat4°Cfor3months.
CyclicAMPConjugateThesuppliedCyclicAMPConjugateConcentrateshouldbediluted1:50withtheConjugateDiluentasindicatedinthetablebelow.
OncedilutedtheCyclicAMPconjugateisstableforonemonthwhenstoredat4°C.1Plate2Plates3Plates4Plates5PlatesConjugateConcentrate50μL100μL150μL200μL250μLConjugateDiluent2.45mL4.9mL7.35mL9.8mL12.25mLFinalMixture2.5mL5mL7.5mL10mL12.5mLChemiluminescentSubstrateMixonepartoftheSubstrateSolutionAwithonepartofSubstrateSolutionBinabrownbottle.
Oncemixedthesubstrateisstableforonemonthwhenstoredat4°C.®www.ArborAssays.com9WEBINSERT150617reagentpreparatiOn-regularfOrmatUsethisformatforurine,salivaandsomecelllysates.
DoNOTuseforplasmasamples.
Allstandardsandsamplesshouldbedilutedinglasstesttubes.
StandardPreparation-regularfOrmatLabeloneglasstesttubeasStock2andfivetubesas#1through#5.
Pipet90μLofSampleDi-luentintotheStock2tubeand450μLofSampleDiluentintotube#1.
Pipet300μLofSampleDiluentintotubes#2to#5.
TheCyclicAMPstocksolutioncontainsanorganicsolvent.
Prerinsethepipettipseveraltimestoensureaccuratedelivery.
Carefullyadd10μLofthecAMPstocksolutiontotheStock2tubeandvortexcompletely.
Take50μLofthecAMPsolutionintheStock2tubeandaddittotube#1andvortexcompletely.
Take150μLofthecAMPsolutionintube#1andaddittotube#2andvortexcompletely.
Repeattheserialdilutionsfortubes#3through#5.
TheconcentrationofCyclicAMPintubes1through5willbe15,5,1.667,0.556,and0.185pmol/mL.
Non-AcetylatedStock2Std1Std2Std3Std4Std5SampleDiluent(μL)90450300300300300AdditionCyclicAMPStd.
Stock2Std1Std2Std3Std4VolofAddition(μL)1050150150150150FinalConc(pM/mL)1501551.6670.5560.185UseStandardswithin1hourofpreparation.®www.ArborAssays.com10WEBINSERT150617aSSayprOtOcOl-regularfOrmat1.
Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.
Determinethenumberofwellstobeusedandreturnunusedwellstothefoilpouchwithdesiccant.
Sealtheziplocplatebagandstoreat4°C.2.
Add50μLofPlatePrimerintoallwellsused.failuretOaddplateprimertOallWellSfirStWillcauSeaSSaytOfail.3.
Pipet75μLSampleDiluentintothenon-specificbinding(NSB)wells.4.
Pipet50μLofSampleDiluentintowellstoactasmaximumbindingwells(B0or0pg/mL).5.
Pipet50μLofsamplesorstandardsintowellsintheplate.
NOTE:SampleDiluentwillturnfromorangetobrightpinkuponsampleorstandardadditiontothePlatePrimerinthewells.6.
Add25μLofthedilutedDetectX®cAMPConjugatetoeachwellusingarepeaterpipet.7.
Add25μLoftheDetectX®cAMPAntibodytoeachwell,excepttheNSBwells,usingarepeaterpipet.8.
Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.
Covertheplatewiththeplatesealerandshakeatroomtemperaturefor2hours.
Iftheplateisnotshaken,signalsboundwillbeapproximately25%lower.9.
Aspiratetheplateandwasheachwell4timeswith300μLwashbuffer.
Taptheplatedryoncleanabsorbenttowels.10.
Add100μLofthemixedChemiluminescentSubstratetoeachwell,usingarepeaterpipet.11.
Incubatetheplateatroomtemperaturefor5minuteswithoutshaking.12.
Readtheluminescencegeneratedfromeachwellinamutimodeorchemiluminescentplatereaderusinga0.1secondreadtimeperwell.
Thechemiluminescentsignalwilldecreaseabout40%over60minutes.13.
Usetheplatereadersbuilt-in4PLCsoftwarecapabilitiestocalculatecAMPconcentrationforeachsample.

SamplePreparation

CellsCelllysisbufferscontaininghighconcentrationsofSDSorotherdetergentsmaynotbecompat-iblewiththisassayormayrequireextradilution.PleasereadInterferentssectiononpage22formoreinformation.Thiskitiscompatiblewitheitheradherentornon-adherentcells.ThecellscanbegrowninanysuitablesterilecontainerssuchasPetridishes,12-,48-or96-wellcultureplatesorflasks.ThecellsmustbeisolatedfromthemediapriortobeinglysedwiththeprovidedSampleDiluent.TheacidicSampleDiluentcontainsdetergentstolysethecells,inactivateendogenousphosphodiesterasesandstabilizethecAMP.Somecelltypesareextremelyhardyandtheendusershouldoptimizethelysisconditionsutilizingfreeze-thawcyclesandultrasonictreatmentstofullylysetheircells.Weused~107JurkatcellspermLofSampleDiluent.Cellnumberneedstobedeterminedbytheendusersinceitwillbedependantoncelltypeandtreatmentconditions.Caremustbetakennottooverdilutethesamples.Foradherentcells,themediashouldbeaspiratedfromthecellsandthecellswashedwithPBS.TheadherentcellsshouldbetreateddirectlywiththeSampleDiluentfor10minutesatroomtemperature.Cellscanbescrapedtodislodgethemfromtheplatesurfaceandcellsshouldbeinspectedtoensurelysis.DetergenthasbeenaddedtotheSampleDiluenttohelplysisoccur.Centrifugethesamplesat≥600xgat4°Cfor15minutesandassaythesupernatantdirectly.Ifrequired,theTCMcanbeassayedforcAMPasoutlinedbelow.Fornon-adherentcells,pelletandwashthecellswithPBSbycentrifugingthesamplesat≥600xgat4°Cfor15minutesasdescribedabove.Treattheaspirated,washedpelletdirectlywiththeSampleDiluentfor10minutesatroomtemperature.Cellsshouldbeinspectedtoensurelysis.DetergenthasbeenaddedtotheSampleDiluenttohelplysisoccur.Centrifugethesamplesat≥600xgat4°Cfor15minutesandassaythesupernatantdirectly.Ifrequired,theTCMcanbeassayedforcAMPasoutlinedbelow.

CalculationofResults

AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
AveragetheduplicateRLUreadingsforeachstandardandsample.
Cre-ateastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanRLUsfortheNSB.
Thesampleconcentrationsobtained,calculatedfromthe%B/B0curve,shouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.
Orusetheonlinetoolfromhttp://www.myassays.com/arbor-assays-cyclic-amp-direct-chemiluminescent-eia-kit-non-acetyl.assaytocalculatethedata.*TheMyAssayslogoisaregisteredtrademarkofMyAssaysLtd.typicaldata-regularfOrmatSampleMeanRLUNetRLU%B/B0CyclicAMPConc.(pmol/mL)NSB19,2400--Standard130,85011,61010.715Standard251,38532,14529.65Standard377,06557,82553.31.667Standard4102,12082,88076.40.556Standard5120,730101,49093.60.185B0127,670108,430100.00Sample142,40523,16521.47.71Sample271,19051,95047.92.14Alwaysrunyourownstandardcurveforcalculationofresults.
Donotusethisdata®www.ArborAssays.com12WEBINSERT150617TypicalStandardCurve-RegularFormat&%&$#$0000<sb@:c="""=""<sb=""!=""1gqzwq="">1]Q^[]Z[:Alwaysrunyourownstandardcurveforcalculationofresults.
Donotusethisdata.
ValidatiOndata-regularfOrmatSensitivityandLimitofDetectionSensitivitywascalculatedbycomparingtheRLUsforeighteenwellsrunforeachoftheB0andstandard#5.
Thedetectionlimitwasdeterminedattwo(2)standarddeviationsfromtheB0alongthestandardcurve.
Sensitivitywasdeterminedas0.119pmol/mL.
Thisisequivalentto5.95fmolcAMPperwell.
TheLimitofDetectionfortheassaywasdeterminedinasimilarmannerbycomparingtheRLUsfortwentyrunsforeachofthezerostandardandalowconcentrationhumanurinesample.
LimitofDetectionwasdeterminedas0.076pmol/mL.
Thisisequivalentto3.8fmolcAMPperwell.®www.ArborAssays.com1300<sb@:c=""web=""insert=""150617=""acetylated=""protocol=""-=""overview=""this=""format=""must=""be=""used=""for=""plasma,=""some=""cell=""lysates=""and=""any=""sample=""with=""low=""camp=""concentrations.=""Priortorunningtheacetylatedassay,allstandards,samplesandtheSampleDiluentusedfortheB0andNSBwellsmustbeacetylated.
Acetylationiscarriedoutbyadding15μLoftheAcetyla-tionReagent(aspreparedbelow)foreach300μLofthestandard,sampleandSampleDiluent.
AfteradditionoftheAcetylationReagentimmediatelyvortexeachtreatedstandard,sampleorSampleDiluentandusewithin30minutesofpreparation.
Note:UponAcetylation,allofthestandardsandsamplesdilutedintheorangeSampleDiluentwillchangetoapaleyellowcolour.reagentpreparatiOn-acetylatedfOrmatAcetylationReagentWorkinginafumehoodmixonepartofAceticAnhydridewith2partsofTriethylamineinaglasstesttube.
UsethefollowingtabletohelpdeterminetheamountofAcetylationReagenttomake.
ReagentsNumberofSamplestobeTested2040100200AceticAnhydrideVolume(μL)2004001,0002,000TriethylamineVolume(μL)4008002,0004,000AcetylationReagentVol(mL)0.61.236UsetheAcetylationReagentwithin60minutesofpreparation.

RestrictionsForResearchUseonly