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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 NAD/NADH Ratio Assay Kit/15273/250 Tests
产品编号:15273
市  场 价:¥100060.00
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:$5003.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Colorimetric NAD/NADH Ratio Assay Kit/15273/250 Tests
商品介绍
Overview
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Ex/Em(nm)460/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellBIOLOGy
CellMetabolism
RelatedRedoxEnzymes
Nicotinamideadeninedinucleotide(NAD+)andnicotinamideadeninedinucleotidephosphate(NADP+)aretwoimportantcofactorsfoundincells.NADHisthereducedformofNAD+,andNAD+istheoxidizedformofNADH.ItformsNADPwiththeadditionofaphosphategrouptothe2"positionoftheadenylnucleotidethroughanesterlinkage.NADPisusedinanabolicbiologicalreactions,suchasfattyacidandnucleicacidsynthesis,whichrequireNADPHasareducingagent.ThetrADItionalNAD/NADHandNADP/NADPHassaysaredonebymonitoringofNADHorNADPHabsorptionat340nm.ThismethodsufferslowsensitivityandhighinterferencesincetheassayisdoneintheUVrangethatrequiresexpensivequartzmicroplate.OurAmplite™NAD/NADHRatioAssayKitprovidesaconvenientmethodforsensitivedetectionofNAD,NADHandtheirratio.TheNADHprobeisachromogenicsensorthathasitsmaximumabsorbanceat~460nmuponNADHreduction.Theabsorbanceincreaseat~460nmisdirectlyproportionaltotheconcentrationofNADHinthesolution.TheNADHprobecanrecognizeNADHinanenzyme-freereaction,andthesignalcanbeeasilyreadbyanabsorbancemicroplatereaderat~460nm.TheAmplite™ColorimetricNADHAssayKitprovidesasensitiveassaytodetectaslittleas3µMNADHina100µLassayvolume.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformat.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADHstocksolution:

Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tohave1mM(1nmol/µL)NADHstocksolution.

Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.PrepareNAD/NADHreactionmixture:

2.1   Add8mLofNADHProbebuffer(ComponentB-II)tothebottleofNAD/NADHRecyclingEnzymeMixture(ComponentA),andmixwell.

2.2   Add2mLNADHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell. 

Note:ThisNAD/NADHreactionmixtureisenoughfor125~200assays.TheunusedNAD/NADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

3.PrepareseriallydilutedNADHstandards(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.

4. RunTotalNAD/NADHAssay(total400assays/kit):

4.1   AddserialdilutionsofNADHstandardandNAD/NADHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.

Note:PreparecellsortissuesamplesasdesiredNAD/NADHLysisBuffer(ComponentG)canbeusedforlysingthecells(Seeappendixfordetails).

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.

 

4.2   Add50μLofNAD/NADHreactionmixture(fromStep2.2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(fromStep4.1)tomakethetotalNAD/NADHassayvolumeof100µL/well.

4.3   Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.

4.4   Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.

5. RunNAD/NADHRatioAssay(total250assays/kit):

5.1   AddseriallydilutedNADHstandardsand/orNAD/NADHcontainingtestsamplesintoawhite/clear96-wellmicroplateasdescribedinTables3and4.

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

Table3.LayoutofNADHstandardsandtestsamplesinawhite/clear96-wellmicroplate

BL

BL

TS

TS

TS(NAD)

TS(NAD)

 

 

 

 

 

 

NS1

NS1

….

….

….

….

 

 

 

 

 

 

NS2

NS2

 

 

 

 

 

 

 

 

 

 

NS3

NS3

 

 

 

 

 

 

 

 

 

 

NS4

NS4

 

 

 

 

 

 

 

 

 

 

NS5

NS5

 

 

 

 

 

 

 

 

 

 

NS6

NS6

 

 

 

 

 

 

 

 

 

 

NS7

NS7

 

 

 

 

 

 

 

 

 

 

Note:NS=NAD/NADHStandards;BL=BlankControl;TS=TestSamples;TS(NAD)=TestSamplestreatedwithNADExtractionSolution(ComponentD)for10to15minutes,thenneutralizedbyNeutralizationSolution(ComponentE).

Table4.Reagentcompositionsforeachwell

NADHStandard

BlankControl

TestSample(NAD/NADH)

TestSample(NADExtract)

SerialDilutions*:25

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