| Assay Method Information | |
| | Assay for In Vitro Inhibitory Activity Against HPK1 Kinase |
| Description: | A kinase buffer (Enzymatic buffer 5×) was diluted to 1×, and 10 mM MgCl2, 1 mM DTT, and 0.005% Tween 20 were added. A 100 ng/μL HPK1 (Life technology) stock solution was diluted with the kinase buffer to obtain a 1.67×, 1.67 ng/μL working solution (final concentration: 1 ng/μL), and the working solution was seeded in a 384-well plate at 6 μL/well. Different compounds dissolved in DMSO were added to the wells using a nanoliter pipettor to obtain final concentrations of the compounds of 1000 nM to 0.244 nM (4-fold serial dilution, 7 concentrations in total), and blank control wells (without enzyme) and negative control wells (with enzyme, plus vehicle DMSO) were set; the wells were set in duplicate. After the enzyme and the compounds were incubated at room temperature for 1 h, a 5×, 5 mM ATP dilution (final concentration: 1 mM) in the kinase buffer was mixed with a 5×, 2.5 M substrate (Cisbio, STK Substrate 1-biotin; final concentration: 500 nM) in equal volume, and the mixture was added to the plate at 4 L/well. The plate was sealed with a film and then incubated at room temperature for 2 h. An assay antibody solution was prepared by mixing the antibody STK Antibody-cryptate (Cisbio, 5 μL/test) and a 4×, 500 nM Streptavidin-XL665 (Cisbio; final concentration: 125 nM) in equal volume and added to the plate at 10 μL/well. The plate was incubated at room temperature for 1 h. The signal values (excitation: 665 nm, emission: 620 nm) were measured using a PE Envision multimode microplate reader, and IC50 was calculated by four-parameter fitting. |
| Affinity data for this assay | |
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