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Ex/Em(nm)	510/524
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	CellBIOLOGy CellMetabolism
Related	RedoxEnzymes 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.Itshouldbestoredat2-8^oCandusedwithin1day. 

 

2.Prepare100XCoAGreen™stocksolution:

Add100µLofDMSO(ComponentD)intothevialofCoAGreen™(ComponentA)tomake100Xstocksolution.

Note:TheunusedCoAGreen™stocksolutionshouldbedividedintosingleusealiquots,storedat-20^oCandkeptfromlight.

 

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

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

 

Table2:Reagentcompositionforeachwell

*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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