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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 NADP/NADPH Ratio Assay Kit/15274/250 Tests
产品编号:15274
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 NADP/NADPH Ratio Assay Kit/15274/250 Tests
商品介绍
Overview
PrinterFriendlyVersion

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™NADP/NADPHRatioAssayKitprovidesaconvenientmethodforsensitivedetectionofNADP,NADPHandtheirratio.TheNADPHprobeisachromogenicsensorthathasitsmaximumabsorbanceat~460nmuponNADHreduction.Theabsorbanceincreaseat~460nmisdirectlyproportionaltotheconcentrationofNADPHinthesolution.TheNADPHprobecanrecognizeNADPHinanenzyme-freereaction,andthesignalcanbeeasilyreadbyanabsorbancemicroplatereaderat~460nm.TheAmplite™ColorimetricNADPHAssayKitprovidesasensitiveassaytodetectaslittleas3µMNADPHina100µLassayvolume.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformat.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADPHstocksolution:

   Add200µLofPBSbufferintothevialofNADPHstandard(ComponentC)tohave1mM(1nmol/µL)NADPHstocksolution.

Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.PrepareNADP/NADPHreactionmixture:

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

2.2   Add2mLNADPHProbe(ComponentB-I)intoabovebottle(fromStep2.1)andmixwell. 

Note:ThisNADP/NADPHreactionmixtureisenoughfor125~200assays.TheunusedNADP/NADPHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

3.PrepareseriallydilutedNADPHstandards(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.

4. RunTotalNADP/NADPHAssay(total400assays/kit):

4.1   AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.

Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentG)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.

 

4.2   Add50μLofNADP/NADPHreactionmixture(fromStep2.2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(fromStep4.1)tomakethetotalNADP/NADPHassayvolumeof100µL/well.

4.3   Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.

4.4   Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.

5. RunNADP/NADPHRatioAssay(total250assays/kit):

5.1   AddseriallydilutedNADPHstandardsand/orNADP/NADPHcontainingtestsamplesintoawhite/clear96-wellmicroplateasdescribedinTables3and4.

Note:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentG)canbeusedforlysingthecellsforconvenience.

Table3.LayoutofNADPHstandardsandtestsamplesinawhite/clear96-wellmicroplate

BL

BL

TS

TS

TS(NADPH)

TS(NADPH)

 

 

 

 

 

 

NS1

NS1

….

….

….

….

 

 

 

 

 

 

NS2

NS2

 

 

 

 

 

 

 

 

 

 

NS3

NS3

 

 

 

 

 

 

 

 

 

 

NS4

NS4

 

 

 

 

 

 

 

 

 

 

NS5

NS5

 

 

 

 

 

 

 

 

 

 

NS6

NS6

 

 

 

 

 

 

 

 

 

 

NS7

NS7

 

 

 

 

 

 

 

 

 

 

Note:NS=NADP/NADPHStandards;BL=BlankControl;TS=TestSamples;TS(NADPH)=TestSamplestreatedwithNADPHExtractionSolution(ComponentD)for15minutes,thenneutralizedbyNeutralizationSolution(ComponentE).

Table4.Reagentcompositionsforeachwell

品牌介绍

AAT Bioquest常用产品报价单

NADPHStandard

BlankControl

TestSample(NADP/NADPH)

TestSample(NADPHExtract)

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