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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*/15276/400 Tests
产品编号:15276
市  场 价:¥85560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*/15276/400 Tests
商品介绍
Overview
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Ex/Em(nm)460/None
MWN/A
CAS#N/A
SolventDMSO
StorageF/D/L
CategoryCellBIOLOGy
CellMetabolism
RelatedRedoxEnzymes
BiochemicalAssays
Nicotinamideadeninedinucleotide(NAD+)andnicotinamideadeninedinucleotidephosphate(NADP+)aretwoimportantcofactorsfoundincells.NADHisthereducedformofNAD+,andNAD+istheoxidizedformofNADH.ItformsNADPwiththeadditionofaphosphategrouptothe2"positionoftheadenylnucleotidethroughanesterlinkage.NADPisusedinanabolicbiologicalreactions,suchasfattyacidandnucleicacidsynthesis,whichrequireNADPHasareducingagent.Inchloroplasts,NADPisanoxidizingagentimportantinthepreliminaryreactionsofphotosynthesis.TheNADPHproducedbyphotosynthesisisthenusedasreducingpowerforthebiosyntheticreactionsintheCalvincycleofphotosynthesis.ThetrADItionalNAD/NADHandNADP/NADPHassaysaredonebymonitoringofNADHorNADPHabsorptionat340nm.ThismethodsufferslowsensitivityandhighinterferencesincetheassayisdoneintheUVrangethatrequiresexpensivequartzmicroplate.ThisAmplite™NADP/NADPHAssayKitprovidesaconvenientmethodforsensitivedetectionofNADPandNADPH.TheenzymesinthesystemspecificallyrecognizeNADP/NADPHinanenzymecyclingreaction.ThereisnoneedtopurifyNADP/NADPHfromsamplemix.Theenzymecyclingreactionsignificantlyincreasesdetectionsensitivity.ComparedtoKit#15260,thiskithashighersensitivity.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADPHstocksolution:

Add200µLofPBSbufferintothevialofNADPHstandard(ComponentC)tomake1mM(1nmol/µL)NADPHstocksolution.

Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.PrepareNADP/NADPHreactionmixture:

2.1   Add8mLofNADPHProbebuffer(ComponentB-II)tothebottleofNADP/NADPHRecyclingEnzymeMixture(ComponentA),andmixwell.

2.2   Add2mLofNADPHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell. 

Note:ThisNADP/NADPHreactionmixtureisenoughfor200assays.TheunusedNADP/NADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

3.PrepareserialdilutionsofNADPHstandard(0to2μM):

3.1   Add2µLof1mMNADPHstocksolution(fromStep1)into998µLPBSbuffer(pH7.4)togenerate2µM(2pmols/µL)NADPHstandardsolution. 

Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.

3.2   Take200µLof2µMNADPHstandardsolution(fromStep3.1)toperform1:2serialdilutionstoget1,0.5,0.25,0.125,0.0625,0.0313and0µMseriallydilutedNADPHstandards.

3.3   AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.

Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience.(Seeappendixfordetails).

Table1:LayoutofNADPHstandardsandtestsamplesinawhite/clearbottom96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

NS1

NS1

….

….

….

….

 

 

 

 

 

 

NS2

NS2

 

 

 

 

 

 

 

 

 

 

NS3

NS3

 

 

 

 

 

 

 

 

 

 

NS4

NS4

 

 

 

 

 

 

 

 

 

 

NS5

NS5

 

 

 

 

 

 

 

 

 

 

NS6

NS6

 

 

 

 

 

 

 

 

 

 

NS7

NS7

 

 

 

 

 

 

 

 

 

 

Note:NS=NADPHStandards,BL=BlankControl,TS=TestSamples.

Table2:Reagentcompositionforeachwell

NADPHStandard

BlankControl

TestSample

SerialDilutions*:50μL

PBS:50μL

50μL

*Note:AddtheseriallydilutedNADPHstandardsfrom0.0313μMto2μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADPH(e.g.,>30μM,finalconcentration)willcausesaturatedsignalandmakethecalibrationcurvenon-linear.

 

4.RunNADPHassayinsupernatantsreaction:

4.1   Add50μLofNADP/NADPHreactionmixture(fromStep2.2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNADP/NADPHassayvolumeof100µL/well

Note1:Fora384-wellplate,add25μLofsampleand25μLofNADP/NADPHreactionmixtureintoeachwell.

Note2:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience.(Seeappendixfordetails).

4.2   Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.

4.3   Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.

References&Citations
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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


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