| Assay Method Information | |
| | Biochemical Assay |
| Description: | The protocol for basic TR-FRET LanthaScreen Tb Kinase Activity Assay inhibitor studies were as follows. LanthaScreen Kinase Activity Assays (ThermoFisher/USA) to evaluate inhibitors were performed by addition of 100 nL of test compound in corresponding DMSO dilutions/5 μL of kinase/fluorescein-ERM(LRRKtide) peptide mixture, 5 μL of ATP into 384 well small volume plates. After incubation for 120 minutes at room temperature, the detection reagents containing Tb-anti-pLRRKtide antibody were added to monitor phosphrylation level of peptide. Then, after 60 min minutes incubation at room temperature plates were read in Envision. Data analysis of emission ratios was according to LanthaScreen Tb Kinase Activity Assay protocol.Kinase and assay components were adjusted to final concentrations according to the kit protocol. For LRRK2: 2 nM wt human LRRK2, catalytic site, catalytic site (ThermoFisher/USA), 400 nM peptide, 38 μM ATP in 1× Kinase Buffer A.Basic protocol for TR-FRET LanthaScreen Tb Kinase Activity Assay inhibitor studies involved two steps:Enzymatic step: Addition of 100 nL of test compound in corresponding DMSO dilutions, 5 μL of kinase/substrate mixture, 5 μL of ATP into 384 well small volume plates. Incubation for 120 minutes at room temperature.Detection step: Addition of 10 μL EDTA & antibody, read plate after 60 minutes. Data analysis of emission ratios according to KinEASE assay protocol.Kinase and assay components were adjusted to final concentrations according to the kit protocol. |
| Affinity data for this assay | |
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