| 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.PrepareNADHstocksolution:
Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tohave1mM(1nmol/µL)NADHstocksolution.
Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNAD/NADHreactionmixture:
Add10mLofNADHSensorBuffer(ComponentB)tothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.
Note:ThisNAD/NADHreactionmixtureisenoughfortwo96-wellplates.TheunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareseriallydilutedNADHstandards(0to10μM):
3.1 Add30µLof1mMNADHstocksolution(fromStep1)into970µLPBSbuffer(pH7.4)togenerate30µM(30pmols/µL)NADHstandardsolution.
Note:DilutedNADHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200µLof30µMNADHstandardsolution(fromStep3.1)toperform1:3serialdilutionstoget10,3,1,0.3,0.1,0.03and0µMseriallydiltuedNADHstandards.
3.3 AddseriallydilutedNADHstandardsand/orNAD/NADHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.
Table1.LayoutofNADHstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | TS(NADH) | TS(NADH) | 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(NADH)=TestSamplestreatedwithNADHExtractionSolutionfor10to15minutes,thenneutralizedbyNADExtractionSolution;TS(NAD)=TestSamplestreatedwithNADExtractionSolutionfor10to15minutes,thenneutralizedbyNADHExtractionSolution.
Table2Reagentcompositionforeachwell
NADHStandard | BlankControl | TestSample(NAD/NADH) | TestSample (NADHExtract) | TestSample (NADExtract) |
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:AddtheseriallydilutedNADHstandardsfrom0.03μMto30μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADH(e.g.,>300μM,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofNADHsensor(toanon-fluorescentproduct).
3.4 ForNADHExtraction(NADH):Add25μLofNADHExtractionSolution(ComponentD)intothewellsofNAD/NADHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,thenadd25μLofNADExtractionSolution(ComponentE)toneutralizetheNADHextractsasdescribedinTables1&2.
ForNADExtraction(NAD):Add25μLofNADExtractionSolution(ComponentE)intothewellsofNAD/NADHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,thenadd25μLofNADHExtractionSolution(ComponentD)toneutralizetheNADextractsasdescribedinTables1&2.
ForTotalNADandNADH:Add25μLof NAD/NADHControlSolution(ComponentF)intothewellsofNADHstandardsandNAD/NADHcontainingtestsamples.Incubateatroomtemperaturefor10to15minutes,andthenadd25μLofControlSolution(ComponentF)asdescribedinTables1and2.
Note1:Preparecellsortissuesamplesasdesired.NAD/NADHLysisBuffer(ComponentG)canbeusedforlysingthecells(Seeappendixfordetails).
Note2:Inhealthymammaliancells,thereismoreNADcomparetoNADH,soonecansimplyusetotalNADandNADHminustheNADtocalculatetheamountofNADH.
4.RunNAD/NADHassayinsupernatantsreaction:
4.1 Add75μLofNADHreactionmixture(fromStep2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(fromStep3.4)tomakethetotalNADHassayvolumeof150µL/well.
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff570nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
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