Meso Scale Discovery- The New Kid on the Block for HTS Protein Kinase Assays (Amgen)

《Meso Scale Discovery- The New Kid on the Block for HTS Protein Kinase Assays (Amgen)》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、MesoScaleDiscovery:TheNewKidontheBlockforHTSProteinKinaseAssaysABeta-SiteEvaluationDavidPowersResearchScientistAmgenInc.ThousandOaks,CAIntroductionMSDContactedAmgenSept’02SignedEarlyAccessProgramFeb‘02Objectives¾Doesitwork?Technology,Hardware,Biology¾WillithaveutilityatAmgen?Integrationi

2、tintoourExistingProcess?Future?Structure¾InstallationandTraining¾Conduct75,000Multi-Arraydetermination(Ser/ThrKinaseScreens)¾Conduct35,000Multi-Spotdeterminations(CytokineSecretagogue)¾ComparisonofMulti-ArrayTechnologyplatformagainstTR-FRET¾ProvideFeedbackMesoScaleSectorHTSCCD12¾PlateBas

3、edA¾ElectrochemiluminescentB¾HomogeneousCounterWorkingDielectric¾ImagingSupportsHighelectrodeelectrodeDensityPlatesSupportHardwareFeatures¾CCDImaging(1min/plate)¾SmallFootprint¾75platecapacityStacker¾StylishDesign¾Morethan3500platescycled¾1500platescycledinthreedays1instrumentfailure(loo

4、sewire)¾NoStackerFailures¾NoCommunicationFailuresWishList¾BarcodePositionOptionsSoftwareFeatures¾StableSoftware¾Simple/IntuitiveGUI¾NoFrills¾PlateIn/DataOutWishList¾AssayBasedProtocol¾ImprovedFileManagementScreeningProtocoloAvidin-Coated28µLBuffer10µLATP+Sub+P+Sub+1AbsAbsWorkingElectrode

5、2µLCompound10µLEnzyme12CounterElectrodeADielectricRT1HourBRu-2oAbo100µLDetecti100µLDetectioon/Stopn+2AbaPhospho-AbBio-PO4SubstrateAvidinReadElectrodeSer/ThrKinaseBiotinylatedSubstrateLabeledGeneric2oAntibodyKinaseAssayDevelopmentMSDKinaseTitration/TimeCourseMSDKinaseDilution4000002500300

6、0002000]2000000.5nMin/MUL1nMULC1500EC1.5nM100000[E2nMteaR10000-100000500-2002040608010012014000.511.522.5Time[min]Enzyme[nM]ATPK[µM]Kinase1MSD-KmforATPmappMesoScaleLanceFBA96.5699.71100.15¾Ser/ThrKinase1/Rate1/R¾TransferableAssaysate00.20.40.6¾ComparableToOtherPlatforms1/[ATP]¾LessEnzyme

7、(5-10fold)02004006008001000[ATP]KinasePreferredMSDKinasePreferred300002500020000LUHI15000LOEC10000500000510152025303540Plate#KinasePreferredHitsConfirmation¾PreferredCollection40µM25¾Kinase=2nM20)15CO¾S/B=40P10t#2(eS¾Z’=0.6850-5-50510152025Set#1(POC)384WellMSD384well10000