| 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:
Add200µLofofPBSbufferintothevialofNADHstandard(ComponentC)tohave1mM(1nmol/µL)NADHstocksolution.
Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20 oC.
2.PrepareNAD/NADHreactionmixture:
Add10mLofNAD/NADHsensorbuffer(ComponentB)intothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.
Note:TheNAD/NADHreactionmixtureisenoughfortwo96-wellplates.TheunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20 oC.
3.PrepareserialdilutionsofNADHstandard(0to10μM):
3.1 Add10μLofNADHstocksolution(fromStep1)into990µLPBSbuffer(pH7.4)togenerate10 µM(10pmol/µL)NADHstandardsolution.
Note:DilutedNADHstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof10μMNADHstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0μMserialdilutionsofNADHstandard.
3.3 AddserialdilutionsofNADHstandardandNAD/NADHcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Preparecellsortissuesamplesasdesired.
Table1LayoutofNADHstandardsandtestsamplesinasolidblack96-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:AddtheseriallydilutedNADHstandardsfrom0.01μMto10μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADH(e.g.,>100μM,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofNADHsensor(toanon-fluorescentproduct).
4.RunNAD/NADHassayinsupernatantsreaction:
4.1 Add50μLofNADHreactionmixture(fromStep2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(fromStep3.3)tomakethetotalNADHassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofNADHreactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm.
Note1:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
Note2:ForNAD/NADHratiomeasurements,kit15263isrecommended.
Note3:ForcellbasedNAD/NADHmeasurements,ReadiUse™mammaliancelllysisbuffer*5X*(cat#20012)isrecommendedtouseforlysingthecells.
| 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
