Deprecated: Required parameter $cat_id follows optional parameter $type in /www/wwwroot/ebimall.com/systems/hong.php on line 2088

Deprecated: Required parameter $where follows optional parameter $tree_id in /www/wwwroot/ebimall.com/systems/hlb.php on line 3505
AntibodiesOnline/Cyclic GMP (cGMP) ELISA Kit/ABIN577673/5 x 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
您好,欢迎您进入188进口试剂采购网网站! 服务热线:4000-520-616
蚂蚁淘商城 | 现货促销 | 科研狗 | 生物在线

AntibodiesOnline/Cyclic GMP (cGMP) ELISA Kit/ABIN577673/5 x 96 tests
Antigen
CyclicGMP(cGMP)
Reactivity
Chemical
Alternatives
Kitswithalternativereactivityto:
21Chemical
2Mouse(Murine)
1Chicken
1Cow(Bovine)
1Dog(Canine)
1General
1Goat
1GuineaPig
1Human
1Monkey
1Pig(Porcine)
1Rabbit
1Rat(Rattus)
1Sheep(Ovine)
MethodType
SandwichELISA
MinimumDetectionLimit
<1 fM cGMP/Sample
Application
ELISA
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®DirectHighSensitivityCyclicGMP(cGMP)ChemiluminescentImmunoassaykitisdesignedtoquantitativelymeasurecGMPpresentinlysedcells,EDTAplasma,urine,salivaandtissueculturemediasamples.
BrandDetectX®
SampleTypeCellLysate,Saliva,Urine,Plasma(EDTA),TissueCultureMedium
AnalyticalMethodQuantitative
DetectionMethodChemiluminescent
SpecificitySpeciesIndependent.SamplesTypesvalidated:CellLysates,Saliva,Urine,EDTAandHeparinPlasma,TissueCultureMedia
Cross-Reactivity(Details)(%)CyclicGMP100 %CyclicAMP<0.1 %GMP<0.1 %AMP<0.1 %ATP<0.1 %
Sensitivity0.034pmol/mL
CharacteristicsTheDirectCyclicGMP(cGMP)ChemiluminescentImmunoassaykitswillmeasurecGMPpresentinlysedcells,EDTAplasma,urine,salivaandculturemediasamples.ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstABIlizers,forcGMPmeasurement.AcidifiedsamplesofcGMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.Afteranovernightincubationat4°C,theplateiswashedandthechemiluminescentsubstrateisadded.Thesubstrategenerateslightwhichisdetectedinamulti-labelmicrotiterplatereadercapableofreADIngluminescence.CyclicGMPisacriticalandmultifunctionalsecondmessengerpresentatlevelstypically10-100foldlowerthancAMPinmosttissues.IntracellularcGMPisformedbytheactionoftheenzymeguanylatecyclaseonGTPanddegradedthroughphosphodiesterasehydrolysis.Guanylatecyclases(GC)areeithersolubleormembranebound.SolubleGCsarenitricoxideresponsive,whereasmembraneboundGCsrespondtohormonessuchasacetylcholine,insulinandoxytocin.OtherchemicalslikeSEROtoninandhistaminealsocauseanincreaseincGMPlevels.CyclicGMPregulatescellularcompositionthroughcGMP-dependentkinase,cGMP-dependentionchannelsortransporters,andthroughitshydrolyticdegradationbyphosphodiesterase.
ComponentsCoatedWhite96WellPlatesAwhiteplasticmicrotiterplate(s)withbreak-apartstripscoatedwithgoatanti-mouseIgG.1Or5Each
CyclicGMPStandard70μLCyclicGMPat640pmol/mLinaspecialstabilizingsolution.
DetectX®CyclicGMPCLIAAntibodyAmousemonoclonalantibodyspecificforcyclicGMP.3mLOr13mL
DetectX®CyclicGMPCLIAConjugateStockAcyclicGMP-peroxidaseconjugateconcentrateinaspecialstabilizingsolution.150μLOr650μL
ConjugateDiluentContainsspecialstabilizersandadditives.3mLOr13mL
SampleDiluentConcentrateA4Xconcentratecontainingstabilizersandadditivesthatmustbedilutedwithdeionizedordistilledwater.CAUSTIC12mLOr60mL
PlatePrimer25mLAneutralizingsolutioncontainingspecialstabilizersandadditives.
AceticAnhydride2mLAceticAnhydrideWARNING:CorrosiveLachrymatorTriethylamine4mLTriethylamineWARNING:CorrosiveLachrymatorWashBufferConcentrateA20Xconcentratethatmustbedilutedwithdeionizedordistilledwater.30mLOr125mL
SubstrateSolutionA6mLOr28mL
SubstrateSolutionB6mLOr28mL
PlateSealer1Or5Each
MaterialnotincludedDistilledordeionizedwater.
Repeaterpipetandtipscapableofdispensing25and100μL.
Glasstesttubes.
Microplateshaker.96wellmicroplatereadercapableofreadingglowchemiluminescence.
Alistofsomemodelsofsuitablereaderscanbefoundonourwebsiteatwww.ArborAssays.com/resources/#general-info.
AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
ThenumberofRLUsobtainedisdependantonthesensitivityandgainofthereaderused.
Ifyouareunsureofhowtoproperlyconfigureyourreadercontactyourplatereadermanufacturerorcarryoutthefollowingprotocol:Dilute5μLoftheCyclicGMPCLIAConjugateConcentrateinto1.495mLofdeionizedwater.
Pipet5μLofdilutedconjugateinto45μLofdeionizedwater.
Pipet5μLofthemixtureintoawhitewellandadd100μLofpreparedCLIAsubstrate(seepage9fordetails).
Thiswellwillgiveyouanintensityslightlyabovethemaximumbindingfortheassay.
Adjustthegainorsensitivitysothatyourreaderisgivingclosetothemaximumsignal.
ToproperlyanalyzethedatasoftwarewillberequiredforconvertingrawRLUreadingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
AlternativeNameCyclicGMP
BackgroundGuanosine3,5-cyclicmonophosphate(cyclicGMP,cGMP)isacriticalandmultifunctionalsec-ondmessengerpresentatlevelstypically10-100foldlowerthancAMPinmosttissues.Intra-cellularcGMPisformedbytheactionoftheenzymeguanylatecyclaseonGTPanddegradedthroughphosphodiesterasehydrolysis1-3.Guanylatecyclases(GC)areeithersolubleormembranebound3,4.SolubleGCsarenitricoxideresponsive,whereasmembraneboundGCsrespondtohor-monessuchasacetylcholine,insulinandoxytocin.OtherchemicalslikeserotoninandhistaminealsocauseanincreaseincGMPlevels5,6.CyclicGMPregulatescellularcompositionthroughcGMP-dependentkinase,cGMP-dependentionchannelsortransporters,andthroughitshydrolyticdeg-radationbyphosphodiesterase1,7
ApplicationNotesThisassayhasbeenvalidatedforlysedcells,saliva,urine,EDTAplasma,tissuesamples,andfortis-sueculturemediasamples.
Samplesshouldbestoredat-70 °Cforlongtermstorage.24-Hoururinesamplesmayneedtohave1 mLconcentratedhydrochloricacidaddedforevery100 mLvolumetoactasapreservative.
SamplescontainingvisIBLeparticulateshouldbecentrifugedpriortousing.
CyclicGMPisidenticalacrossallspeciesandweexpectthiskitmaymeasurecGMPfromsourcesotherthanhuman.
TheendusershouldevaluaterecoveriesofcGMPinothersamplesbeingtested.
AfterdilutionintheSampleDiluent(seepage9)theremaybesomeprecipitationofproteinsandthesupernatantfromthecentrifugedsamplesshouldbeused.
AfterbeingdilutedinSampleDiluentthesamplescanbeassayeddirectlywithin2hours,orfrozenat≤-70 °Cforlateranalysis.
Severelyhemolyzedsamplesshouldnotbeusedinthiskit.
ForsamplescontaininglowlevelsofcGMP,theacetylatedassayprotocolmustbeusedduetoitsenhancedsensitivity.
Allstandardsandsamplesshouldbedilutedinglasstesttubes.
Comment

Samplevalues:Fourhumanplasmasamplesweretestedintheassay.
Sampleswerediluted10-20foldandrunintheassay.
Valuesrangedfrom3.0to8.0pmol/mLwithanaverageforthesamplesof5.48pmol/mL.
Fivenormalhumanurinesamplesweredilutedbetween50and2,000foldinSampleDiluentandvaluesrangedintheneatsamplesfrom44.2to564pmol/mLwithanaverageforthesamplesof287.2pmol/mL

PlatePre-coated
ProtocolForsampleswherethelevelsofcGMPareexpectedtoberelativelyhigh,theregularformatfortheassaycanbeused.
ForsampleswithexpectedlowlevelsofcGMP,anoptionalacetylationprotocolcanbeused.
ThekitisuniqueinthatallsamplesandstandardsaredilutedintoanacidicSampleDiluent,whichcontainsspecialadditivesandstabilizers,forcGMPmeasurement.
Thisallowsplasma,urineandsalivasamplestobereadinanidenticalmannertolysedcells.
AcidifiedsamplesofcGMParestableandendogenousphosphodiesterasesareinactivatedintheSampleDiluent.
AcGMPstandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.
AwhitemicrotiterplatecoatedwithanantibodytocapturemouseIgGisprovidedandaneutralizingPlatePrimersolutionisaddedtoalltheusedwells.
Standardsordilutedsamples,eitherwithorwithoutacetylation,arePipettedintotheprimedwells.
AcGMP-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.
ThebindingreactionisinitiatedbytheadditionofamousemonoclonalantibodytocGMPtoeachwell.
Afteranover-nightincubationat4 °C,theplateiswashedandthechemiluminescentsubstrateisadded.
ThesubstratereactswiththeboundcGMP-peroxidaseconjugatetoproducelightThegeneratedlightisdetectedinamicrotiterplatereadercapableofreadingluminescence.
TheconcentrationofthecGMPinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders.
ReagentPreparation

Allowthekitreagentstothawandcometoroomtemperaturefor30-60minutes.
WerecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydeterminecGMPconcentrations.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.
WashBufferDiluteWashBufferConcentrate1:20byaddingonepartoftheconcentratetonineteenpartsofdeionizedwater.
Oncedilutedthisisstableatroomtemperaturefor3months.
SampleDiluentPreparetheSampleDiluentbydilutingtheSampleDiluentConcentrate1:4,addingonepartoftheconcentratetothreepartsofdeionizedwater.
Oncedilutedthisisstableat4°Cfor3months.
CyclicGMPConjugateThesuppliedCyclicGMPCLIAConjugateConcentrateshouldbediluted1:20withtheConjugateDiluentasindicatedinthetablebelow.
OncedilutedtheCyclicGMPconjugateisstableforonemonthwhenstoredat4°C.1Plate2Plates3Plates4Plates5PlatesConjugateConcentrate125μL250μL375μL500μL625μLConjugateDiluent2.375mL4.75mL7.125mL9.5mL11.875mLFinalMixture2.5mL5mL7.5mL10mL12.5mLChemiluminescentSubstrateMixonepartoftheSubstrateSolutionAwithonepartofSubstrateSolutionBinabrownbottle.
Oncemixedthesubstrateisstableforonemonthwhenstoredat4°C.®www.ArborAssays.com9reagentpreparatiOn-regularfOrmatAllstandardsandsamplesshouldbedilutedinglasstesttubes.
StandardPreparation-regularfOrmatLabeloneglasstesttubeasStock2andseventubesas#1through#7.
Pipet150μLofSampleDiluentintotheStock2tubeand296μLofSampleDiluentintotube#1.
Pipet150μLofSampleDiluentintotubes#2to#7.
TheCyclicGMPstocksolutioncontainsanorganicsolvent.
Prerinsethepipettipseveraltimestoensureaccuratedelivery.
Carefullyadd10μLofthecGMPstocksolutiontotheStock2tubeandvortexcompletely.
Take24μLofthecGMPsolutionintheStock2tubeandaddittotube#1andvortexcompletely.
Take150μLofthecGMPsolutionintube#1andaddittotube#2andvortexcompletely.
Repeattheserialdilutionsfortubes#3through#7.
TheconcentrationofCyclicGMPintubes1through7willbe3,1.5,0.75,0.375,0.188,0.094,and0.047pmol/mL.
Non-AcetylatedStock2Std1Std2Std3Std4Std5Std6Std7SampleDiluent(μL)150296150150150150150150AdditionCyclicStockStd1Std2Std3Std4Std5Std6GMPStd.2VolofAddition(μL)1024150150150150150150FinalConc(pM/mL)4031.50.750.3750.18750.09380.0469UseStandardswithin1hourofpreparation.®www.ArborAssays.com10aSSayprOtOcOl-regularfOrmat1.
Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.
Determinethenumberofwellstobeusedandreturnunusedwellstothefoilpouchwithdesiccant.
Sealtheziplocplatebagandstoreat4°C.2.
Add50μLofPlatePrimerintoallwellsused.failuretOaddplateprimertOallWellSfirStWillcauSeaSSaytOfail.3.
Pipet75μLSampleDiluentintothenon-specificbinding(NSB)wells.4.
Pipet50μLofSampleDiluentintowellstoactasmaximumbindingwells(B0or0pmol/mL).5.
Pipet50μLofsamplesorstandardsintowellsintheplate.
NOTE:SampleDiluentwillturnfromorangetobrightpinkuponsampleorstandardadditiontothePlatePrimerinthewells.6.
Add25μLofthedilutedDetectX®cGMPCLIAConjugatetoeachwellusingarepeaterpipet.7.
Add25μLoftheDetectX®cGMPCLIAAntibodytoeachwell,excepttheNSBwells,usingarepeaterpipet.8.
Covertheplatewiththeplatesealerandshaketheplatefor15minutesatroomtemperature.9.
Placethecoveredplateina4°Crefrigeratorfor16hours.10.
ThefollowingdayremovetheChemiluminescentSubstratefromtherefrigeratorandallowtocometoroomtemperatureforatleast30minutes.
AdditionofcoldSubstratewillcausedepressedsignal.11.
Taketheplatefromtherefrigeratorandwasheachwell4timeswith300μLwashbuffer.
Taptheplatedryoncleanabsorbenttowels.12.
Add100μLofthemixedChemiluminescentSubstratetoeachwell,usingarepeaterpipet.13.
Incubatetheplateatroomtemperaturefor5minuteswithoutshaking.14.
Readtheluminescencegeneratedfromeachwellinamutimodeorchemiluminescentplatereaderusinga0.1secondreadtimeperwell.
Thechemiluminescentsignalwilldecreaseabout40%over60minutes.15.
Usetheplatereadersbuilt-in4PLCsoftwarecapabilitiestocalculatecGMPconcentrationforeachsample.

SamplePreparation

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

CalculationofResults

AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
AveragetheduplicateRLUreadingsforeachstandardandsample.
Cre-ateastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanRLUsfortheNSB.
Thesampleconcentrationsobtained,calculatedfromthe%B/B0curve,shouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.
Orusetheonlinetoolfromhttp://www.myassays.com/arbor-assays-cyclic-gmp-direct-chemiluminescent-kit-non-acetyl.assaytocalculatethedata.*TheMyAssayslogoisaregisteredtrademarkofMyAssaysLtd.typicaldata-regularfOrmatSampleMeanRLUNetRLU%B/B0CyclicGMPConc.(pmol/mL)NSB12,9450--Standard122,5309,58513.523Standard229,60516,66023.511.5Standard337,48024,53534.620.75Standard448,80535,86050.600.375Standard560,93047,98567.700.1875Standard671,46558,52082.570.0938Standard776,60063,65589.810.0469B083,82070,8751000Sample126,65513,71019.341.86Sample240,69527,75039.150.62Alwaysrunyourownstandardcurveforcalculationofresults.®www.ArborAssays.com12TypicalStandardCurve-RegularFormat10070,000908060,0007050,0006040,00050%B/B0NetRLU4030,0003020,0002010,00010000.010.1110cGMPConc.(pmol/mL)Alwaysrunyourownstandardcurveforcalculationofresults.
Donotusethisdata.
ValidatiOndata-regularfOrmatSensitivityandLimitofDetectionSensitivitywascalculatedbycomparingtheRLUsfortwentywellsrunforeachoftheB0andstandard#7.
Thesensitivitywasdeterminedattwo(2)standarddeviationsfromtheB0alongthestandardcurve.
Sensitivitywasdeterminedas0.034pmol/mL.
TheLimitofDetectionfortheassaywasdeterminedinasimilarmannerbycomparingtheRLUsfortwentyrunsforeachofthezerostandardandalowconcentrationhumanurinesample.
LimitofDetectionwasdeterminedas0.047pmol/mL®www.ArborAssays.com13%B/B0NetRLUacetylatedprOtOcOl-OVerVieWUsethisformatforanysamplewithlowcGMPconcentrations.
Priortorunningtheacetylatedassay,allstandards,samplesandtheSampleDiluentusedfortheB0andNSBwellsmustbeacetylated.
Acetylationiscarriedoutbyadding10μLoftheAcetyla-tionReagent(aspreparedbelow)foreach200μLofthestandard,sampleandSampleDiluent.
Immediatelyvortexeachtreatedstandard,sampleorSampleDiluentafteradditionoftheAcetyla-tionReagentandusewithin30minutesofpreparation.
Note:UponAcetylation,allofthestandardsandsamplesdilutedintheorangeSampleDiluentwillchangetoapaleyellowcolour.reagentpreparatiOn-acetylatedfOrmatAcetylationReagentWorkinginafumehoodmixonepartofAceticAnhydridewith2partsofTriethylamineinaglasstesttube.
UsethefollowingtabletohelpdeterminetheamountofAcetylationReagenttomake.
ReagentsNumberofSamplestobeTested2040100200AceticAnhydrideVolume(μL)2004001,0002,000TriethylamineVolume(μL)4008002,0004,000AcetylationReagentVol(mL)0.61.236UsetheAcetylationReagentwithin60minutesofpreparation.®www.ArborAssays.com14StandardPreparation-AcetylatedFormatAllstandardsandsamplesshouldbedilutedinglasstesttubes.
LabeloneglasstesttubeasStock2andsixtubesas#1through#6.
Pipet150μLofSampleDiluentintotheStock2tubeand585μLofSampleDiluentintotube#1.
Pipet300μLofSampleDiluentintotubes#2to#6.
TheCyclicGMPstocksolutioncontainsanorganicsolvent.
Prerinsethepipettipseveraltimestoensureaccuratedelivery.
Carefullyadd10μLofthecGMPstocksolutiontotheStock2tubeandvortexcompletely.
Take15μLofthecGMPsolutionintheStock2tubeandaddittotube#1andvortexcompletely.
Take300μLofthecGMPsolutionintube#1andaddittotube#2andvortexcompletely.
Repeattheserialdilutionsfortubes#3through#6.
TheconcentrationofCyclicGMPintubes1through6willbe1,0.5,0.25,0.125,0.0625,and0.0313pmol/mL.
AcetylatedStock2Std1Std2Std3Std4Std5Std6SampleDiluent(μL)150585300300300300300AdditionCyclicStock2Std1Std2Std3Std4Std5GMPStd.
VolofAddition(μL)1015300300300300300FinalConc(pM/mL)4010.50.250.1250.06250.03125StandardandSampleAcetylationPipet300μLofSampleDiluentintoaglasstubetoactastheZerostandard/NSBtube.
Add15μLofAcetylationReagenttothistubeandvorteximmediately.
Proceedtoassaywithin30minutes.
Pipet200μLofeachstandardorsampletobetestedintofreshglasstubes.
Add10μLoftheAcetylationReagentintoeachtubeandvorteximmediately.
Proceedtoassaywithin30minutes.
NOTE:SamplesandSampleDiluentwillturnfromorangetopaleyellowuponacetylation.
UseAcetylatedStandardsandSampleswithin30minutesofpreparation.

RestrictionsForResearchUseonly
PrecautionofUse•Aswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlabo-ratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.•Theantibodycoatedplateneedstobestoreddesiccated.
Thesilicagelpackincludedinthefoilziplocbagwillkeeptheplatedry.
Thesilicagelpackwillturnfrombluetopinkiftheziplochasnotbeenclosedproperly•Thiskitutilizesaperoxidase-basedreadoutsystem.
Buffers,includingothermanufacturersWashBuffers,containingsodiumazide

新闻动态
行业前沿
技术文章
最新产品