| Assay Method Information | |
| | HPK1 ADP-Glo Enzyme Assay |
| Description: | The buffer used in the enzyme assay contains 5 mM MOPS (pH=7.2), 2.5 mM β-Glycerol Phosphate, 0.4 mM EDTA, 1 mM EGTA, 0.05 mM DTT, 5 mM MgCl2. The compound was dissolved in 100% DMSO at the concentration was 10 mM. The initial concentration of the test was 5 uM, and ten data points were diluted by three-fold gradient, and each point was repeatedly measured twice. The HPK1 protein was purchased from Thermo (Cat. No. PV6355) and diluted to 2× stock solution at the concentration of 10 nM (the final concentration of the enzyme assay was 5 nM). 2.5 l of 2×HPK1 protein was added to each well of the plate containing the test compound, centrifuged at 1000 rpm for 30 seconds, and then incubated at 25° C. for 15 minutes. MBP protein was purchased from Millipore (Cat. No. 13-110) and ATP was purchased from Sigma (Cat. No. A7699-5G) and the two were formulated into 2× working solutions at concentrations of 4 uM and 80 uM. Added 2.5 μl mixture of 2×MBP and ATP, centrifuged at 1000 rpm for 30 seconds, then incubated at 25° C. for 90 minutes. Then added 5 ul of ADP-Glo (Promega, Cat. No. V9102) to the assay plate and incubated at 1000 rpm for 30 minutes at 25° C. for 60 minutes. Finally, 10 ul of the kinase assay reagent (Promega, Cat. No. V9102) was added to the assay plate, centrifuged at 1000 rpm for 30 seconds at 25° C. for 60 minutes, and the fluorescence intensity was determined. |
| Affinity data for this assay | |
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