| 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)tohave1mM(1nmol/µL)NADPHstocksolution.
Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNADP/NADPHreactionmixture:
Add10mLofNADPHSensorBuffer(ComponentB)intothebottleofNADP/NADPHRecyclingEnzymeMixture(ComponentA),andmixwell.
Note:ThisNADP/NADPHreactionmixtureisenoughfortwo96-wellplates.TheunusedNADP/NADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofNADPHstandard(0to10μM):
3.1 Add10µLof1mMNADPHstocksolution(fromStep1)to990µLPBSbuffer(pH7.4)togenerate10 µM(10pmols/µL)NADPHstandardsolution.
Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200µLof10µMNADPHstandardsolution(fromStep3.1)toperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0µMserialdilutionsofNADPHstandard.
3.3 AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.NADP/NADPHLysisBuffer(ComponentG)canbeusedforlysingthecellsforconvenience(Seeappendixfordetails).
Table1.LayoutofNADPHstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | TS(NADPH) | TS(NADHP) | TS(NADP) | TS(NADP) |
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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=NADP/NADPHStandards;BL=BlankControl;TS=TestSamples;TS(NADPH)=TestSamplestreatedwithNADPHExtractionSolutionfor10to15minutes,thenneutralizedbyNADPExtractionSolution;TS(NADP)=TestSamplestreatedwithNADPExtractionSolutionfor10to15minutes,thenneutralizedbyNADPHExtractionSolution.
Table2Reagentcompositionforeachwell
NADPHStandard | BlankControl | TestSample(NADP/NADPH) | TestSample(NADPHExtract) | TestSample (NADPExtract) | ||||
SerialDilutions*:25μL | PBS:25μL | TestSample:25μL | TestSample:25μL | TestSample:25μL | ||||
ComponentF:25μL | ComponentF:25μL | ComponentF:25μL | ComponentD:25μL | ComponentE:25μL | ||||
Incubateatroomtemperaturefor10to15minutes | ||||||||
ComponentF:25μL | ComponentF:25μL | ComponentF:25μL | ComponentE:25μL | ComponentD:25μL | ||||
Total:75μL | Total:75μL | Total:75μL | Total:75μL | Total:75μL | ||||
*Note:AddtheseriallydilutedNADPHstandardsfrom0.01μMto3μMintowellsfromNS1toNS7induplicate.High
concentrationofNADPH(e.g.,>100μM,finalconcentration)maycausereducedfluorescencesignalduetotheover
oxidationofNADPHsensor(toanon-fluorescentproduct).
3.4 ForNADPHExtraction(NADPH):Add25μLofNADPHExtractionSolution(ComponentD)intothewellsofNADP/NADPHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,thenadd25μLofNADPExtractionSolution(ComponentE)toneutralizetheNADPHextractsasdescribedinTables1&2.
ForNADPExtraction(NADP):Add25μLofNADPExtractionSolution(ComponentE)intothewellsofNADP/NADPHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,thenadd25 μLofNADPHExtractionSolution(ComponentD)toneutralizetheNADPextractsasdescribedinTables1&2.
ForTotalNAPDandNADPH:Add25μLofNADP/NADPHControlSolution(ComponentF)intothewellsofNADPHstandardsandNADP/NADPHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,
andthenadd25μLofControlSolution(ComponentF)asdescribedinTables1&2.
Note1:Preparecellsortissuesamplesasdesired.NADP/NADPHLysisBuffer(ComponentG)canbeusedforlysingthecells(Seeappendixfordetails).
Note2:Inhealthymammaliancells,thereismoreNADPHcomparetoNADP,soonecansimplyusetotalNADPandNADPHminustheNADPHtocalculatetheamountofNADP.
4.RunNADP/NADPHassayinsupernatantsreaction:
4.1 Add75μLofNADPHreactionmixture(fromStep2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(fromStep3.4)tomakethetotalNADPHassayvolumeof150µL/well.
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm.
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