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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™比色总NADP和NADPH分析试剂盒/15260/400试验
商品详细AAT Bioquest/Amplite™比色总NADP和NADPH分析试剂盒/15260/400试验
AAT Bioquest/Amplite™比色总NADP和NADPH分析试剂盒/15260/400试验
AAT Bioquest/Amplite™比色总NADP和NADPH分析试剂盒/15260/400试验
商品编号: 15260
品牌: aatbio
市场价: ¥85560.00
美元价: 51336.00
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)575/None
MWN/A
CAS#N/A
SolventN/A
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.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADPHstocksolution:

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

Note:TheunusedNADPHstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

2.PrepareNADP/NADPHreactionmixture:

Add10mLofNADP/NADPHSensorBuffer(ComponentB)intothebottleofNADP/NADPHRecyclingEnzymeMixture(ComponentA),andmixwell.

Note:ThisNADP/NADPHreactionmixtureisenoughfortwo96-wellplates.TheunusedNADP/NADPHmixtureshouldbedividedintosingleusealiquotsandstoredat-20oC.

 

3.PrepareserialdilutionsofNADPHstandard(0to10μM):

3.1   Add10μLofNADPHstocksolution(fromStep1)to990µLPBSbuffertogenerate10µM(10pmol/µL)NADPHstandardsolution.

Note:DilutedNADPHstandardsolutionisunstable,andshouldbeusedwithin4hours.

 

3.2   Take200μLof10μMNADPHstandardsolutiontoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01,0.003and0μMserialdilutionsofNADPHstandard.

 

3.3   AddserialdilutionsofNADPHstandardandNADP/NADPHcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2

Note:Preparecellsortissuesamplesasdesired.

Table1LayoutofNADPHstandardsandtestsamplesinawhite/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:AddtheseriallydilutedNADPHstandardsfrom0.003μMto3μMintowellsfromNS1toNS7induplicate.HighconcentrationofNADPH(e.g.,>100μM,finalconcentration)maycausereducedsignalduetotheoveroxidationofNADPHsensor.

 

4.RunNADP/NADPHassayinsupernatants:

4.1   Add50μLofNADPHreactionmixture(fromStep2)intoeachwellofNADPHstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalNADPHassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofNADPHreactionmixtureintoeachwell.

 

4.2   Incubatethereactionatroomtemperaturefor15minutesto2hours,protectedfromlight.

 

4.3   Monitortheabsorbanceincreasewithanabsorbanceplatereaderat575±5nmorattheabsorbanceratioof~570nmto~605nmtoincreaseassaysensitivity.

Note1:ForNADP/NADPHratiomeasurements,kit15263isrecommended.

Note2:ForcellbasedNADP/NADPHmeasurements,ReadiUse™mammaliancelllysisbuffer*5X*(cat#20012)isrecommendedtouseforlysingthecells.

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Journal:JournalofBiologicalChemistry(2016):7621--7636

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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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