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AAT Bioquest/Amplite™ Colorimetric Total NAD and NADH Assay Kit/15258/400 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareNADHstocksolution:
Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tohave1mM(1nmol/µL)NADHstocksolution.
Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNAD/NADHreactionmixture:
Add10mLofNAD/NADHSensorBuffer(ComponentB)tothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.
Note:Thissolutionisenoughfortwo96-wellplates.AnyunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofNADHstandard(0to10μM):
3.1 Add10μLofNADHstocksolution(fromStep1)into990µLPBSbuffer(pH7.4)togenerate10 µMdilutedNADHstandardsolution.
Note:DilutedNADHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof10μMstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0 μMserialdilutionsofNADHstandard.
3.3 AddserialdilutionsofNADHstandardandNAD/NADHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.
Table1LayoutofNADHstandardsandtestsamplesinawhite/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.
Table2Reagentcompositionforeachwell
NADHStandard | BlankControl | TestSample |
SerialDilutions*:50μL | PBS:50μL | 50μL |
Note:*AddtheseriallydilutedNADHstandardsfrom0.01μMto10μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADH(e.g.,>100μM,finalconcentration)maycausereducedsignalduetotheoveroxidationofNADHsensor.
4.RunNAD/NADHassayinsupernatantsreaction:
4.1 Add50μLofNADHreactionmixture(fromStep2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(fromStep3.3)tomakethetotalNADHassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofNADHreactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat575±5nmorattheabsorbanceratioof~570nmto~605nmtoincreaseassaysensitivity.
Note1:ForNAD/NADHratiomeasurements,kit15263isrecommended.
Note2:ForcellbasedNAD/NADHmeasurements,ReadiUse™mammaliancelllysisbuffer*5X*(cat#20012)isrecommendedtouseforlysingthecells.
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