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Ex/Em(nm)	571/585
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection ProteinKinases
Related	CellSignalingMolecules 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.="">

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparesamples:

1.1   Thawallthesixcomponentsatroomtemperaturebeforeuse.

 

1.2   AvoiddirectexposureofADPSensorI(ComponentB1)tolight.

Note:AliquotandstoretheunusedADPSensorBuffer(ComponentA)and50xADPSensorIstocksolution(from3.1)at-20^oC.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.RunAmplite™ADPassay:

Warning:TheADPassayshouldberunatpHfrom6.5to7.4.

3.1 Make50XADPSensorIstocksolutionbyadding50uLDMSO(ComponentB3)intovialofADPSensor1(ComponentB1).

Note:Aliquotunused50XADPSensor1DMSOstocksolution,storeat-20^oC,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

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