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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™通用荧光激酶分析试剂盒*红色荧光*/31002/500测试
商品详细AAT Bioquest/Amplite™通用荧光激酶分析试剂盒*红色荧光*/31002/500测试
AAT Bioquest/Amplite™通用荧光激酶分析试剂盒*红色荧光*/31002/500测试
AAT Bioquest/Amplite™通用荧光激酶分析试剂盒*红色荧光*/31002/500测试
商品编号: 31002
品牌: aatbio
市场价: ¥9900.00
美元价: 5940.00
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
Overview
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Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
ProteinKinases
RelatedCellSignalingMolecules
BiochemicalAssays
MostofcommercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.ForthekinaseassaykitsthatarebasedondetectionofphosphopeptidesonehastospendtimeandeffortstoidentifyanoptimizedpeptidesubstratewhiletheATPdepletionmethodsuffersvariousinterferencesduetotheuseofluciferasethatareinhibitedoractivatedbyvariousBIOLOGicalcompounds.TheAmplite™UniversalKinaseAssayKitisbasedonthemonitoringofADPformation,whichisdirectlyproportionaltoenzymephoshphotransferaseactivityandismeasuredfluorimetricaly.Thiskitprovidesafast,simple,andhomogeneousassayformeasurekinasesactivities.Thecharacteristicsofitshighsensitivity(<0.2 um="" adp),="" broad="" atp="" tolerance="" (1-300="" um),="" non-antibody="" based,="" non-radioactive="" and="" no-wash="" method="" to="" detect="" the="" amount="" of="" adp="" produced="" as="" a="" result="" of="" enzyme="" activity="" make="" it="" an="" ideal="" kit="" for="" determining="" kinase="" michaelis-menten="" kinetics="" and="" for="" screening="" and="" identifying="" kinase="" inhibitors.="" the="" assay="" can="" be="" performed="" in="" a="" convenient="" 96-well="" or="" 384-well="" microtiter-plate="" format="" and="" easily="" adapted="" to="" automation="" with="" no="" separation="" steps="" required.="">
SpectrumAdvancedSpectrumViewer



Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparesamples:

1.1   Thawallthesixcomponentsatroomtemperaturebeforeuse.

 

1.2   AvoiddirectexposureofADPSensorI(ComponentB1)tolight.

Note:AliquotandstoretheunusedADPSensorBuffer(ComponentA)and50xADPSensorIstocksolution(from3.1)at-20oC.Avoidrepeatedfreeze/thawcyclesandpotentialADPcontaminationfromexogenousbiologicalsources.

 

1.3Blackplatesarestronglyrecommendedtoachievethebestresults.

 

2.Runkinasereaction(Reagentsarenotprovidedforthisstep):

Warning:TheADPSensorisunstableinthepresenceofthiolssuchasDTTand-mercaptoethanol.Finalthiolconcentrationhigherthan10μMwouldsignificantlydecreasetheassaydynamicrange.

2.1   Prepare20µL(or10µLfor384-wellplate)ofkinasereactionsolution/wellasdesired.Thecomponentsofkinasereactionshouldbeoptimizedasneeded(e.g.,anoptimizedbuffersystemmightberequiredforaspecifickinasereaction).

 

2.2   Inmostcases,ADPassaybuffer(ComponentD)canalsobeusedtorunkinasereactionifyoudonothavetheoptimizedkinasebuffer.

 

2.3   TheAmplite™FluorimetricKinaseAssayKitisusedtodeterminetheADPformation.

 

3.RunAmpliteADPassay:

Warning:TheADPassayshouldberunatpHfrom6.5to7.4.

3.1 Make50XADPSensorIstocksolutionbyadding50uLDMSO(ComponentB3)intovialofADPSensor1(ComponentB1).

Note:Aliquotunused50XADPSensor1DMSOstocksolution,storeat-20oC,protectfromlight

3.2   MakeADPSensorbyadding50uLof50XADPSensorIstocksolution(fromStep3.1)intovialofADPSensorII(ComponentB2).

Note:ThereconstitutedADPsensorisnotstable,makefreshasneeded.

 

3.3   Add20µL(or10µLfor384-wellplate)ofADPSensorBuffer(ComponentA)and10µL(or10µLfor384-wellplate)ofADPSensor(fromStep3.2)intoeachwellfilledwiththe20µL(or10µLfor384-wellplate)kinasereactionsolution(seeStep2.1)tomakethetotalADPassayvolumeof50µL/well(or25µLfor384-wellplate).

 

3.4   Incubatethereactionmixtureatroomtemperaturefor15minutesto1hour.

 

3.5   MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm.

 

4.GenerateanADPcalibrationcurve(Notrequiredforthescreeningofkinaseinhibitors):

Note:AnADPstandardcurvecanbegeneratedasdescribedbelow.

 

4.1   Add100µLofddH2OintoADPStandard(ComponentC)tomakea300mMADPstocksolution.MakeserialdilutionsofADPstandardinthekinasereactionbufferbyincludingasamplewithoutADPformeasuringbackgroundfluorescence.

Note:Typically,ADPconcentrationsrangingfrom0.05to30μMareappropriate.

 

4.2   AddthesameamountoftheseriallydilutedADPstandardsintoanemptyplate(20µL/wellfora96-wellplate,10µL/wellfora384-wellplate).

 

4.3   Add20µL(fora96-wellplate)or10µL(fora384-wellplate)/wellofADPSensorBuffer(ComponentA)and10µL(fora96-wellplate)or5µL(fora384-wellplate)ofADPSensor(fromStep3.2)intoeachwellofseriallydilutedADPstandards(fromStep4.2)tomakethetotalvolumeof50µL(fora96-wellplate)or25µL(fora384-wellplate)foreachreaction.

 

4.4   Incubatethereactionmixtureatroomtemperaturefor15minutesto1hour.

 

4.5   MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm(Cutoff570nm).

 

4.6   GenerateanADPstandardcurve.

References&Citations
CitationExplorer

DiscoveryofNon-ATP-CompetitiveInhibitorsofPolo-likeKinase1
Authors:TaikangxiangYun,TanQin,YingLiu,LuhuaLai
Journal:ChemMedChem(2016):713--717

CellpolaritykinaseMST4cooperateswithcAMP-dependentkinasetoorchestratehistamine-stimulatedacidsecretioningastricparietalcells
Authors:HaoJiang,WenwenWang,YinZhang,WilliamWYao,JiyingJiang,BoQin,WendyYYao,FushengLiu,HuihuiWu,TarshaLWard
Journal:JournalofBiologicalChemistry(2015):28272--28285

RegulationofNDR1activitybyPLK1ensuresproperspindleorientationinmitosis
Authors:MaomaoYan,LingluoChu,BoQin,ZhikaiWang,XingLiu,ChangjiangJin,GuanglanZhang,MartaGomez,AlexanderHergovich,ZhengjunChen
Journal:Scientificreports(2015):10449

Trypanosomabruceivacuolartransporterchaperone4(TbVtc4)isanacidocalcisomepolyphosphatekinaserequiredforinvivoinfection
Authors:NoeliaLander,PaulNUlrich,RobertoDocampo
Journal:JournalofBiologicalChemistry(2013):34205--34216


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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