| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 460/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes |
1.PrepareNADPHstocksolution:
Add200µLofPBSbufferintothevialofNADPHstandard(ComponentC)tomake1mM(1nmol/µL)NADHstocksolution.
Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNADPHreactionmixture:
Add1mLofNADPHProbe(ComponentA)into4mLNADPHAssayBuffer(ComponentB),andmixwell.
Note:5mLNADPHreactionmixtureisforone96-well.TheunusedNADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofNADPHstandard(0to100μM):
3.1 Add100μLofNADPHstocksolution(fromStep1)into400µLPBSbuffer(pH7.4)togenerate200 µM(100pmol/µL)NADPHstandardsolution. Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof200μMNADPHstandardsolutiontoperform1:2serialdilutionstoget100,50,25,12.5,6.25,3.13and0μMserialdilutionsofNADPHstandard.
AddserialdilutionsofNADPHstandardandNADPHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience(Seeappendixfordetails).
Table1LayoutofNADPHstandardsandtestsamplesinawhite/clearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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NS1 | NS1 | …. | …. | …. | …. |
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NS2 | NS2 |
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NS3 | NS3 |
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NS4 | NS4 |
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NS5 | NS5 |
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NS6 | NS6 |
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NS7 | NS7 |
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Note:NS=NADPHStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
NADPHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | PBS:50μL | 50μL |
*Note:AddtheseriallydilutedNADPHstandardsfrom3.13μMto200μMintowellsfromNS1toNS7induplicate.
4.RunNADPHassayinsupernatantsreaction:
4.1 Add50μLofNADPHreactionmixture(fromStep2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNADPHassayvolumeof100µL/well
Note1:Fora384-wellplate,add25μLofsampleand25μLofNADPHreactionmixtureintoeachwell.
Note2:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience(Seeappendixfordetails).
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.
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