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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/PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*/21655/100 Tests
产品编号:21655
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/PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*/21655/100 Tests
商品介绍
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Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
Anions
RelatedCellSignalingMolecules
ProteinKinases
ADPisinvolvedinmanyBIOLOGicalreactionssuchasproteinkinases.OurADPassaykitcanbeusedforassayingproteinkinasereactionsuniversallybymonitoringADPformation,whichisdirectlyproportionaltoenzymephosphotransferaseactivityandismeasuredfluorimetrically.Thiskitprovidesafast,simpleandhomogeneousassayformeasuringADPformationordepletion.Theassayiscontinuous,andcanbeeasilyadaptedtoautomation.Thekitisconvenient,requiringminimalhands-ontime.Proteinkinasesareofinteresttoresearchersinvolvedindrugdiscoveryduetotheirbroadrelevancetodiseasessuchascancerandotherproliferativediseases,inflammatorydiseases,metabolicdisordersandneurologicaldiseases.MostofcommercialproteinkinaseassaykitsareeitherbasedonmonitoringofphosphopeptideformationorATPdeletion.OurADPassaykitismorerobustforassayingproteinkinasesthanmostofcommercialkinaseassaykits.
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   Prepare20µLofkinasereactionsolutionasdesired.Thecomponentsofkinasereactionshouldbeoptimizedasneeded(e.g.,anoptimizedbuffersystemmightberequiredforaspecifickinasereaction).

 

2.2   Inmostcases,ADPassaybuffer(ComponentD)canalsobeusedtorunkinasereactionifyoudonothavetheoptimizedkinasebuffer.

 

2.3   TheAmplite™FluorimetricKinaseAssayKitisusedtodeterminetheADPformation.

 

3.RunAmpliteADPassay:

Warning:TheADPassayshouldberunatpHfrom6.5to7.4.

3.1 Make50XADPSensorIstocksolutionbyadding20uLDMSO(ComponentB3)intovialofADPSensorI(ComponentB1).

Note:Aliquotunused50XADPSensorIDMSOstocksolution,storeat-20oC,protectfromlight

3.2   MakeADPSensorbyadding20uLof50XADPSensorIstocksolution(fromStep3.1)intovialofADPSensorII(ComponentB2).

3.3   Add20µLofADPSensorBuffer(ComponentA)and10µLofADPSensor(fromStep3.2)intoeachwellfilledwiththe20µLkinasereactionsolution(seeStep2)tomakethetotalADPassayvolumeof50µL/well.

 

3.4   Incubatethereactionmixtureatroomtemperaturefor15minutesto1hour.

 

3.5   MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm.

 

4.GenerateanADPcalibrationcurve(Notrequiredforthescreeningofkinaseinhibitors):

Note:AnADPstandardcurvecanbegeneratedasdescribedbelow.

 

4.1   Add100µLofH2OintoADPStandard(ComponentC)tomakea300mMADPstocksolution.MakeserialdilutionsofADPstandardinthekinasereactionbufferbyincludingasamplewithoutADPformeasuringbackgroundfluorescence.

Note:Typically,ADPconcentrationsrangingfrom0.05to30μMareappropriate.

 

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

 

4.3   Add20µLofADPSensorBuffer(ComponentA)and10µLofADPSensor(fromStep3.2)intoeachwellofseriallydilutedADPstandards(fromStep4.2)tomakethetotalvolumeof50µLforeachreaction.

 

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


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