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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric Coenzyme A Quantitation Kit *Green Fluorescence*/15270/200 Tests
产品编号:15270
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AAT Bioquest/Amplite™ Fluorimetric Coenzyme A Quantitation Kit *Green Fluorescence*/15270/200 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)510/524
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellBIOLOGy
CellMetabolism
RelatedRedoxEnzymes
BiochemicalAssays
CoenzymeA(CoA)isauniversalandessentialcofactorinallformsofcellularlifeactingasaprincipalacylcarrierinnumerousbiosynthetic,energy-yielding,anddegradativepathways.Itplaysimportantrolesinthesynthesisandoxidationoffattyacids,pyruvateoxidationandthecitricacidcycle.MeasurementofCoAisoneoftheessentialtasksforinvestigatingbiologicalprocessesandeventsinmanybiologicalsystems.ThereareafewreagentsorassaykitsavailableforquantitatingCoAcontentinbiologicalsystems.However,theexistingcommercialkitseitherlacksensitivityorhavetediousprocedures.OurAmplite™FluorimetricCoAQutitationAssayKitprovidesanultrasensitivefluorimetricassaytoquantitateCoAcontentbydetectionof-SHgroupinCoA.OurproprietaryfluorogenicCoAGreen™dyeusedinthekitbecomesstronglyfluorescentuponreactingwith-SH.Theassaykitcandetectaslittleas4picomoleofCoAina100µLassayvolume(40nM).Itcanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatatEx/Em=490/520nm,andeasilyadaptedtoautomationwithoutaseparationstep.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareCoAstandardstocksolution:

Add200µLofddH2OintotheCoAstandardvial(ComponentC)tomake1mM(1nmol/µL)stocksolution.

Note1:ItishighlyrecommendedtousetheddH2OthathasbeenspargedwithnitrogentoremoveoxygenforpreparingcoenzymeAstocksolution.

Note2:Theaqueoussolutionisnotstable,willdegraderapidly.Itshouldbestoredat2-8oCandusedwithin1day. 

 

2.Prepare100XCoAGreen™stocksolution:

Add100µLofDMSO(ComponentD)intothevialofCoAGreen™(ComponentA)tomake100Xstocksolution.

Note:TheunusedCoAGreen™stocksolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.

 

3.PrepareCoAAssaymixture:

Add50µLof100XCoAGreen™stocksolution(fromStep2)into5mLofAssayBuffer(ComponentB),andmixthemwell.

 

4.PrepareserialdilutionsofCoAstandard(0to30μM):

4.1   Add30μLofCoAstandardstocksolution(fromStep1)to970µLofAssayBuffer(ComponentB)togenerate30µM(30pmol/µL)CoAstandard.

Note:DilutedCoAstandardsolutionisunstable,andshouldbeusedwithin4hours.

4.2   Take200μLof30μMCoAstandardsolutiontoperform1:3serialdilutionswithAssaybuffer(ComponentB)toget10,3,1,0.3,0.1,0.03,0.01and0μMserialdilutionsofCoAstandard.

4.3   AddCoAstandardsandCoA-containingtestsamplesintoasolidblack96-wellmicroplateasshowninTables1and2.

Note:Treatcellsortissuesamplesasdesired.

Table1:LayoutofCoAstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

CoA1

CoA1

….

….

….

….

 

 

 

 

 

 

CoA2

CoA2

 

 

 

 

 

 

 

 

 

 

CoA3

CoA3

 

 

 

 

 

 

 

 

 

 

CoA4

CoA4

 

 

 

 

 

 

 

 

 

 

CoA5

CoA5

 

 

 

 

 

 

 

 

 

 

CoA6

CoA6

 

 

 

 

 

 

 

 

 

 

CoA7

CoA7

 

 

 

 

 

 

 

 

 

 

Note:CoA=CoAStandards,BL=BlankControl,TS=TestSamples.

 

Table2:Reagentcompositionforeachwell

CoAStandard

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer:50μL

50μL

*Note:AddtheserialdilutionsofCoAstandardfrom0.01μMto10μMintowellsfromCoA1toCoA7induplicate.

 

5.RunCoAassay:

5.1   Add50μLofCoAreactionmixture(fromStep3.1)toeachwelloftheCoAstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalCoAassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofCoAreactionmixtureintoeachwell.

5.2   Incubatethereactionatroomtemperaturefor10minutesto1hour,protectedfromlight.

5.3   MonitorthefluorescenceincreaseatEx/Em=490/520nmwithafluorescenceplatereader.

References&Citations
CitationExplorer

MethodsformeasuringCoAandCoAderivativesinbiologicalsamples
Authors:YugoTsuchiya,UyenPham,IvanGout
Journal:BiochemicalSocietyTransactions(2014):1107--1111


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