| 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.PrepareNADHstocksolution:
Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tohave1mM(1nmol/µL)NADHstocksolution.
Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNAD/NADHreactionmixture:
2.1 Add8mLofNADHProbebuffer(ComponentB-II)tothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.
2.2 Add2mLNADHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell.
Note:ThisNAD/NADHreactionmixtureisenoughfor125~200assays.TheunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareseriallydilutedNADHstandards(0to10μM):
3.1 Add10µLof1mMNADHstocksolution(fromStep1)into990µLPBSbuffer(pH7.4)togenerate10µM(10pmols/µL)NADHstandardsolution.
Note:DilutedNADHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200µLof10µMNADHstandardsolution(fromStep3.1)toperform1:2serialdilutionstoget5,2.5,1.25,0.625,0.313,0.156,0.078and0µMseriallydilutedNADHstandards.
4. RunTotalNAD/NADHAssay(total400assays/kit):
4.1 AddserialdilutionsofNADHstandardandNAD/NADHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.
Note:PreparecellsortissuesamplesasdesiredNAD/NADHLysisBuffer(ComponentG)canbeusedforlysingthecells(Seeappendixfordetails).
Table1.LayoutofNADHstandardsandtestsamplesinawhite/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=NADHStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
NADHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | PBS:50μL | 50μL |
*Note:AddtheseriallydilutedNADHstandardsfrom0.078μMto5μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADH(e.g.,>100μM,finalconcentration)willcausesaturatedsignalandmakethecalibrationcurvenon-linear.
4.2 Add50μLofNAD/NADHreactionmixture(fromStep2.2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(fromStep4.1)tomakethetotalNAD/NADHassayvolumeof100µL/well.
4.3 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.4 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.
5. RunNAD/NADHRatioAssay(total250assays/kit):
5.1 AddseriallydilutedNADHstandardsand/orNAD/NADHcontainingtestsamplesintoawhite/clear96-wellmicroplateasdescribedinTables3and4.
Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentG)canbeusedforlysingthecellsforconvenience(Seeappendixfordetails).
Table3.LayoutofNADHstandardsandtestsamplesinawhite/clear96-wellmicroplate
BL | BL | TS | TS | TS(NAD) | TS(NAD) |
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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=NAD/NADHStandards;BL=BlankControl;TS=TestSamples;TS(NAD)=TestSamplestreatedwithNADExtractionSolution(ComponentD)for10to15minutes,thenneutralizedbyNeutralizationSolution(ComponentE).
Table4.Reagentcompositionsforeachwell
NADHStandard | BlankControl | TestSample(NAD/NADH) | TestSample(NADExtract) |
SerialDilutions*:25 |
