| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 510/524 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
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 | …. | …. |
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CoA1 | CoA1 | …. | …. | …. | …. |
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CoA2 | CoA2 |
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CoA3 | CoA3 |
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CoA4 | CoA4 |
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CoA5 | CoA5 |
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CoA6 | CoA6 |
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CoA7 | CoA7 |
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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
