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AAT Bioquest/PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*/21655/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection Anions |
| Related | CellSignalingMolecules ProteinKinases |
| Spectrum | AdvancedSpectrumViewer |
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.RunAmplite™ADPassay:
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
