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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™ Colorimetric NADH Assay Kit/15271/400 Tests
商品详细AAT Bioquest/Amplite™ Colorimetric NADH Assay Kit/15271/400 Tests
AAT Bioquest/Amplite™ Colorimetric NADH Assay Kit/15271/400 Tests
AAT Bioquest/Amplite™ Colorimetric NADH Assay Kit/15271/400 Tests
商品编号: 15271
品牌: aatbio
市场价: ¥71060.00
美元价: 42636.00
产地: 美国(厂家直采)
公司:
产品分类: 其它检测试剂盒
公司分类: Other_kits
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍
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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+.NADformsNADPwiththeadditionofaphosphategrouptothe2"positionoftheadenylnucleotidethroughanesterlinkage.ThetrADItionalNAD/NADHandNADP/NADPHassaysarebasedonmonitoringthechangesinNADHorNADPHabsorptionat340nm.TheshortUVwavelengthofNAD/NADHandNADP/NADPHassaysmakesthetraditionalmethodssufferlowsensitivityandhighinterference.DuetotheweakabsorptionofNADandNADH,theUVabsorptionmethodrequireslargesamplesizes,makingNADandNADHmeasurementsunpracticalforlimitedsamplesize.AATBioquest"sAmplite™ColorimetricNADHAssayKitprovidesaconvenientmethodforthedetectionofNADH.TheNADHprobeisachromogenicsensorthathasitsmaximumabsorbanceat460nmuponNADHreduction.Theabsorbanceincreaseat460nmisdirectlyproportionaltotheconcentrationofNADHinthesolution.TheNADHprobecanrecognizeNADHinanenzyme-freereaction,andthesignalcanbeeasilyreadbyanabsorbancemicroplatereaderat~460nm.TheAmplite™ColorimetricNADHAssayKitprovidesasensitiveassaytodetectaslittleas3µMNADHina100µLassayvolume.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformat.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADHstocksolution:

Add200µLofPBSbufferintothevialofNADHstandard(ComponentC)tomake1mM(1nmol/µL)NADHstocksolution.

Note:TheunusedNADHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

2.PrepareNADHreactionmixture:

Add1mLofNADHProbe(ComponentA)into4mLNADHAssayBuffer(ComponentB),andmixwell.

Note:5mLNADHreactionmixtureisforone96-well.TheunusedNADHreactionmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

3.PrepareserialdilutionsofNADHstandard(0to100μM):

3.1   Add100μLofNADHstocksolution(fromStep1)into400µLPBSbuffer(pH7.4)togenerate200 µM(100pmol/µL)NADHstandardsolution. Note:DilutedNADHstandardsolutionisunstable,andshouldbeusedwithin4hours.

3.2   Take200μLof200μMNADHstandardsolutiontoperform1:2serialdilutionstoget100,50,25,12.5,6.25,3.13and0μMserialdilutionsofNADHstandard.

3.3   AddserialdilutionsofNADHstandardandNADHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.Note:Preparecellsortissuesamplesasdesired.

Table1LayoutofNADHstandardsandtestsamplesinawhite/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.

Table2Reagentcompositionforeachwell

NADHStandard

BlankControl

TestSample

SerialDilutions*:50μL

PBS:50μL

50μL

*Note:AddtheseriallydilutedNADHstandardsfrom3.13μMto200μMintowellsfromNS1toNS7induplicate.

 

4.RunNADHassayinsupernatantsreaction:

4.1   Add50μLofNADHreactionmixture(fromStep2)intoeachwellofNADHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNADHassayvolumeof100µL/well

Note1:Fora384-wellplate,add25μLofsampleand25μLofNADHreactionmixtureintoeachwell.

Note2:Preparecellsortissuesamplesasdesired.LysisBuffer(ComponentD)canbeusedforlysingthecellsforconvenience.

 (Seeappendixfordetails).

4.2   Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.

4.3   Monitortheabsorbanceincreasewithanabsorbanceplatereaderat460nm.

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品牌介绍
美国AAT Bioquest Inc.(前身是ABD Bioquest,Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域,包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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