| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 340/430 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes |
1.Prepareseriallydilutedascorbicacidstandards(0to1mM)andtestsamples:
1.1 Add200μLofddH2Ointoascorbicacidstandardvial(ComponentD)tomake100mMascorbicacidstandardsolution.
Note:AscorbicacidaqueoussolutionisnotstableandcanbeoxidizedtoDHA.Pleasestoreunusedascorbicacidat-20oC,avoidlightandrepeatedfreeze-thawcycles.
1.2 Prepareascorbicacidstandarddilutions:Add10μLof100mMascorbicacid(fromstep1.1)into990μLofassaybuffertoget1mMascorbicacidsolution.Take300μLof1mMascorbicacidstandardsolutioninto600μLassaybuffertoperform1:3serialdilutionstoget300,100,30,10,3,and1μMseriallydilutedascorbicacidstandards.
1.3 Addascorbicacidcontainingsamplesandseriallydilutedascorbicacidstandardsintoasolidblack96-wellmicroplateaccordingtoTables1.
Table1:Layoutofascorbicacidstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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AA1 | AA1 | …. | …. | …. | …. |
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AA2 | AA2 |
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AA3 | AA3 |
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AA4 | AA4 |
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AA5 | AA5 |
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AA6 | AA6 |
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AA7 | AA7 |
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Note1:AA=AscorbicAcidStandard,BL=BlankControl(assaybuffer),TS=TestSample.
Note2:AddtheserialdilutionsofAscorbicAcidstandardfrom1μMto1000μMintowellsfromAA1toAA7.
2.Prepareascorbicacidassaymixture:
2.1 MakeAscorbrite™Bluestocksolution:Add50μLofDMSO(ComponentE)intoAscorbrite™Blue(ComponentA)tomake200XAscorbrite™Bluestocksolution.
Note:Storeunused200XAscorbrite™Bluestocksolutionat-20oC,avoidlightandrepeatedfreeze-thawcycles.
2.2 MakeascorbicacidassaymixtureaccordingtoTables2:
Table2:AscorbicAcidAssayMixtureforone96-wellplate:
Components | TotalAAAssay | DHAAssay |
AssayBuffer(ComponentC) | 5mL | 5mL |
EnzymeMix(ComponentB) | 1bottle | None |
Ascorbrite™BluestockSolution(200X,fromStep2.1) | 25μL | 25μL |
TotalVolume | 5.025mL | 5.025mL |
Note1:Theassaymixtureisnotstable,useitpromptly,andavoiddirectexposuretolight.
Note2:Onecanmakeenzymestocksolutionbyadding100μLddH2OintooneEnzymeMixbottle(ComponentB)tomake50Xenzymestocksolution,anduseitproportionallyfortotalAAassay(forexample,for1mLtotalAAassaymixture,add20μL50Xenzymestocksolutionand5μL200XAscorbrite™BluestockSolutioninto1mLAssayBuffer).Storeunused100Xenzymestocksolutionat-20oC,avoidrepeatedfreeze-thawcycles.
3.Runtotalascorbicacidassay:
3.1 Add50μLofAssayMixture(fromStep2.3)intoeachwellofascorbicacidstandard,blankcontrol,andtestsamples(seeStep1.3)tomakethetotalascorbicacidassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsample,25μLofAssaymixtureintoeachwell.
3.2 Incubatethereactionmixtureatroomtemperaturefor30minutesto1hour.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=340/430nm(cutoff:420nm).
| References&Citations |  PrinterFriendlyVersion |
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