| 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)tohave1mM(1nmol/µL)NADPHstocksolution.
Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNADP/NADPHreactionmixture:
2.1 Add8mLofNADPHProbebuffer(ComponentB-II)tothebottleofNADP/NADPHRecyclingEnzymeMixture(ComponentA),andmixwell.
2.2 Add2mLNADPHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell.
Note:ThisNADP/NADPHreactionmixtureisenoughfor125~200assays.TheunusedNADP/NADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareseriallydilutedNADPHstandards(0to2μM):
3.1 Add2µLof1mMNADPHstocksolution(fromStep1)into998µLPBSbuffer(pH7.4)togenerate2µM(2pmols/µL)NADPHstandardsolution.
Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200µLof2µMNADPHstandardsolution(fromStep3.1)toperform1:2serialdilutionstoget1,0.5,0.25,0.125,0.0625,0.0313and0µMseriallydilutedNADPHstandards.
4. RunTotalNADP/NADPHAssay(total400assays/kit):
4.1 AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentG)canbeusedforlysingthecellsforconvenience.(Seeappendixfordetails).
Table1.LayoutofNADPHstandardsandtestsamplesinawhite/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.
Table2.Reagentcompositionforeachwell
NADPHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | PBS:50μL | 50μL |
*Note:AddtheseriallydilutedNADPHstandardsfrom0.0313μMto2μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADPH(e.g.,>30μM,finalconcentration)willcausesaturatedsignalandmakethecalibrationcurvenon-linear.
4.2 Add50μLofNADP/NADPHreactionmixture(fromStep2.2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(fromStep4.1)tomakethetotalNADP/NADPHassayvolumeof100µL/well.
4.3 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.4 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.
5. RunNADP/NADPHRatioAssay(total250assays/kit):
5.1 AddseriallydilutedNADPHstandardsand/orNADP/NADPHcontainingtestsamplesintoawhite/clear96-wellmicroplateasdescribedinTables3and4.
Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentG)canbeusedforlysingthecellsforconvenience.
Table3.LayoutofNADPHstandardsandtestsamplesinawhite/clear96-wellmicroplate
BL | BL | TS | TS | TS(NADPH) | TS(NADPH) |
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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)=TestSamplestreatedwithNADPHExtractionSolution(ComponentD)for15minutes,thenneutralizedbyNeutralizationSolution(ComponentE).
Table4.Reagentcompositionsforeachwell
NADPHStandard | BlankControl | TestSample(NADP/NADPH) | TestSample(NADPHExtract) |
