| Assay Method Information | |
| | Pharmacological Activity Assay |
| Description: | The luminescent signal generated is proportional to the ADP concentration produced in a kinase assay in the presence and absence of the compound(s) and is correlated with the kinase activity. Two microlitres (μl) of enzyme mix consisting of 10 nM HPK1, 30 nM GLK, or 2 nM LCK in 1× reaction buffer (50 mM HEPES (pH7.2), 1 mM DL-Dithiothreitol (DTT), 0.005% (vol/vol) Brij35, 20 mM MgCl2) was spotted into Greiner 384-well low volume plate is with 0.1 μl of compound, which was dosed at 100, 31.25, 12.5, 3.19, 1, 0.32, 0.1, 0.032, 0.01 and 0.003 μM of final test concentrations and preincubated for 30 minutes at room temperature. Enzymatic reactions were initiated with 2 μl of peptide substrate/ATP mix (for HPK1: 10 μM LRRKtide (RLGRDKYKTLRQIRQ-amide; Cambridge Research Biochemicals, Billingham, UK), 30 μM ATP; for GLK: 14 μM LRRKtide, 60 μM ATP; for LCK: 100 μM LCKtide (EQEDEDEPEGIYGVLE-amide; Intonation, Boston, MA), 50 μM ATP) in 1× reaction buffer and incubated at room temperature for 60 minutes. 4 μl ADP-Glo Reagent was added to terminate the reactions and deplete the remaining ATP and incubated at room temperature for 60 minutes. Finally, 8 μl ADP-Glo Max Detection Reagent was added to simultaneously convert ADP to ATP and incubated at room temperature for 60 minutes. The newly synthesized ATP is converted to light using a luciferase/luciferin reaction. Luminescence was read by PHERAstar FSX plate reader (BMG LABTECH, Cary, NC) and the data was captured by PHERAstar FSX MARS data analysis software. |
| Affinity data for this assay | |
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