| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 460/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareNADHstocksolution:Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tomake1mM(1nmol/µL)NADHstocksolution.Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
2.PrepareNAD/NADHreactionmixture:
2.1 Add8mLofNADHProbebuffer(ComponentB-II)tothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.
2.2 Add2mLofNADHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell.
Note:ThisNAD/NADHreactionmixtureisenoughfor200assays.TheunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.PrepareserialdilutionsofNADHstandard(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.
3.3 AddserialdilutionsofNADHstandardandNAD/NADHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.Note:Preparecellsortissuesamplesasdesired.
Table1:LayoutofNADHstandardsandtestsamplesinawhite/clearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
NS1 | NS1 | …. | …. | …. | …. |
|
|
|
|
|
|
NS2 | NS2 |
|
|
|
|
|
|
|
|
|
|
NS3 | NS3 |
|
|
|
|
|
|
|
|
|
|
NS4 | NS4 |
|
|
|
|
|
|
|
|
|
|
NS5 | NS5 |
|
|
|
|
|
|
|
|
|
|
NS6 | NS6 |
|
|
|
|
|
|
|
|
|
|
NS7 | NS7 |
|
|
|
|
|
|
|
|
|
|
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.RunNADHassayinsupernatantsreaction:
4.1 Add50μLofNAD/NADHreactionmixture(fromStep2.2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNAD/NADHassayvolumeof100µL/well
Note1:Fora384-wellplate,add25μLofsampleand25μLofNADHreactionmixtureintoeachwell.
Note2:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience.
(Seeappendixfordetails).
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.
| References&Citations |  CitationExplorer |
CelastrolattenuatesangiotensinIImediatedhumanumbilicalveinendothelialcellsdamagethroughactivationofNrf2/ERK1/2/Nox2signalpathway
Authors:MiaoLi,XinLiu,YongpengHe,QingyinZheng,MinWang,YuWu,YuanpengZhang,ChaoyunWang
Journal:EuropeanJournalofPharmacology(2017):124--133
CytosolicRedoxStatusofWineYeast(SaccharomycesCerevisiae)underHyperosmoticStressduringIcewineFermentation
Authors:FeiYang,CaitlinHeit,DebraLInglis
Journal:Fermentation(2017):61
EpigeneticregulationofRunx2transcriptionandosteoblastdifferentiationbynicotinamidephosphoribosyltransferase
Authors:MinLing,PeixinHuang,ShamimaIslam,DanielPHeruth,XuananLi,LiQinZhang,Ding-YouLi,ZhaohuiHu,ShuiQingYe
Journal:Cell&Bioscience(2017):27
MCU-dependentmitochondrialCa2+inhibitsNAD+/SIRT3/SOD2pathwaytopromoteROSproductionandmetastasisofHCCcells
Authors:TRen,HZhang,JWang,JZhu,MJin,YWu,XGuo,LJi,QHuang,HYang
Journal:Oncogene(2017)
Metabolicandmolecularinsightsintoanessentialroleofnicotinamidephosphoribosyltransferase
Authors:LiQZhang,LeonVanHaandel,MinXiong,PeixinHuang,DanielPHeruth,CharlieBi,RogerGaedigk,XunJiang,Ding-YouLi,GeraldWyckoff
Journal:CellDeath&Disease(2017):e2705
PyrroloquinolineQuinone,aRedox-activeo-Quinone,StimulatesMitochondrialBiogenesisbyActivatingSIRT1/PGC-1αSignalingPathway
Authors:KazuhiroSaihara,RyosukeKamikubo,KazutoIkemoto,KojiUchida,MitsuguAkagawa
Journal:Biochemistry(2017)
ResveratrolattenuatesexcessiveethanolexposureinducedinsulinresistanceinratsviaimprovingNAD+/NADHratio
Authors:GangLuo,BingqingHuang,XiangQiu,LinXiao,NingWang,QinGao,WeiYang,LipingHao
Journal:MolecularNutrition&FoodResearch(2017)
ASnapshotofthePlantGlycatedProteomeSTRUCTURAL,FUNCTIONAL,ANDMECHANISTICASPECTS
Authors:TatianaBilova,ElenaLukasheva,DominicBrauch,UtaGreifenhagen,GaganPaudel,ElenaTarakhovskaya,NadezhdaFrolova,JulianeMittasch,GerdUlrichBalcke,AlainTissier
Journal:JournalofBiologicalChemistry(2016):7621--7636
AMPKactivationprotectscellsfromoxidativestress-inducedsenescenceviaautophagicfluxrestorationandintracellularNAD+elevation
Authors:XiaojuanHan,HaoranTai,XiaoboWang,ZheWang,JiaoZhou,XiaweiWei,YiDing,HuiGong,ChunfenMo,JieZhang
Journal:Agingcell(2016):416--427
Cell-LineSelectivityImprovesthePredictivePowerofPharmacogenomicAnalysesandHelpsIdentifyNADPHasBioMarkerforFerroptosisSensitivity
Authors:KenichiShimada,MikiHayano,NenCPagano,BrentRStockwell
Journal:Cellchemicalbiology(2016):225--235
