| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 422/466 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareNADPstocksolution:
Add500µLofddH2OintothevialofNADPstandard(ComponentD)tomake1mMNADPstocksolution.
Note:TheunusedNADPstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareserialdilutionsofNADPstandard(0to10μM):
2.1 Add10μLofNADPstocksolution(fromStep1)into990µLH2OorPBSbuffertogenerate10 µMNADPstandardsolution.
Note:DilutedNADPstandardsolutionisunstable,andshouldbeusedwithin4hours.
2.2 Take200μLof10μMNADPstandardsolutiontoperform1:3serialdilutionsinH2OorPBStoget10,3,1,0.3,0.1,0.03,0.01,and0μMserialdilutionsofNADPstandard.
2.3 Add50μLserialdilutionsofNADPstandardandNADPcontainingtestsamplesintoablack/solidbottom96-wellmicroplateasdescribedinTable1:
Table1LayoutofNADPstandardsandtestsamplesinablack/solidbottom96-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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Note1:NS=NADPStandards,BL=BlankControl,TS=TestSamples.
Note2:AddtheseriallydilutedNADPstandardsfrom10μMto0.01μMintowellsfromNS1toNS7induplicate.
3.RunNADPassay:
3.1 Add20μLQuestFluor™NADPProbe(ComponentA)solutionintoeachwellofNADPstandard,blankcontrol,andtestsamples,mixwell.
3.2 Add20μLAssaySolution(ComponentB)intoeachwell,mixwell.
Note:Fora384-wellplate,add25μLofsampleand10μLofQuestFluor™NADPProbeand10μLAssaySolutionintoeachwell.
3.3 Incubatethereactionatroomtemperaturefor10-20minutes,protectedfromlight.
3.4 Add15μLEnhancer(ComponentC)toeachwelltomakethetotalNADPassayvolumeof105µL/well,andincubateatroomtemperaturefor10-20minutes,protectedfromlight.
Note:Fora384-wellplate,add7.5uLEnhancer.
3.5 Monitorthefluorescenceincreasewithafluorescenceplatereaderat420/480nm.
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