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| | HPK1 Kinase Assay (Biochemical Assay) |
| Description: | A recombinant fusion protein consisting of full-length human HPK1 (MAP4K1) with an N-terminal Glutatione S-transferase (GST) tag was produced in insect cells Sf21 using the baculovirus expression system. GST-HPK1 protein was purified from cell lysates by glutathione Sepharose affinity chromatography. The assay is run in three continuous steps: 1) the HPK1 enzymatic kinase reaction, 2) an ATP depletion, and 3) the ADP detection, the steps 2 and 3 are performed with ADP-Glo Kinase Assay kit from Promega (V9101). Test compounds were prepared by 10-point serial dilution in dimethyl sulfoxide (DMSO) and 100 nL of each dilution was spotted onto a 384-well Optiplate (Perkin Elmer and Cat #6007299) by Labcyte Echo. 5 μL kinase reaction buffer (0.02% Brij-35, 2 mM DTT, 50 mM HEPES pH 7.5, MgCl2 10 mM, BSA 0.01% and O-glycerophosphate 12.5 mM) containing HPK1 (3.2 nM) enzyme was transferred to each well and incubated for 15 minutes at room temperature at 60% humidity. The enzymatic reaction was started by adding 5 μl of Start-Mix (10 μM ATP and 3.235 μM MBP). After 120 minutes, the reaction was stopped by adding 5 μL of ADP-Glo reagent (Promega, V9101) and incubating for 40 min. in the dark at 23 C. To determine the level of ADP, 10 μl ADP-Glo Detection solution was added and incubated for 1 hour at 23 C. in the dark. The plate was transferred to a Perkin Elmer EnVision (2104 Multilabel Reader) for luminescence detection and percent inhibition activity and IC50 value were determined using Genedata Screener. |
| Affinity data for this assay | |
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