| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareNADPHstocksolution:
Add200µLofPBSbufferintothevialofNADPHStandard(ComponentC)tomake1mM(1nmol/µL)NADPHstocksolution.
Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNADP/NADPHreactionmixture:
Add10mLofNADP/NADPHSensorBuffer(ComponentB)intothebottleofNADP/NADPHRecyclingEnzymeMixture(ComponentA),andmixwell.
Note:ThisNADP/NADPHreactionmixtureisenoughfortwo96-wellplates.TheunusedNADP/NADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofNADPHstandard(0to10μM):
3.1 Add10μLofNADPHstocksolution(fromStep1)to990µLPBSbuffertogenerate10µM(10 pmol/µL)NADPHstandardsolution.
Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof10μMNADPHstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01,0.003and0μMserialdilutionsofNADPHstandard.
3.3 AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.
Table1.LayoutofNADPHstandardsandtestsamplesinasolidblack96-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.
Table2.Reagentcompositionforeachwell
NADPHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | PBS:50μL | 50μL |
*Note:AddtheseriallydilutedNADPHstandardsfrom0.003μMto3μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADPH(e.g.,>100μM,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofNADPHsensor(toanon-fluorescentproduct).
4.RunNADP/NADPHassayinsupernatants:
4.1 Add50μLofNADPHreactionmixture(fromStep2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNADPHassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofNADPHreactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalat540/590nm).
Note1:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
Note2:ForNADP/NADPHratiomeasurements,kit15264isrecommended.
Note3:ForcellbasedNADP/NADPHmeasurements,ReadiUse™mammaliancelllysisbuffer*5X*(cat#20012)isrecommendedtouseforlysingthecells.
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