| Assay Method Information | |
| | Biological Assay |
| Description: | Table A: Primary Assay used to determine potency of HPK1 enzymatic activity inhibition. Compound activity was determined using recombinant HPK1 protein and MBP Substrate (both Promega, Cat #V6398) in an in vitro enzymatic reaction. The enzymatic assay used to determine activity was a Luminescence assay using a Microplate Reader ClarioStar Plus. The enzymatic reaction was carried out in assay buffer (40 mM TRIS-HCl pH 7.4-7.6, 20 mM MgCl2, 0.05 mM DTT, 0.1 mg/ml BSA). The compounds were dispensed on a 384 well Diamond Well Plate (Axigen, Cat #P-384-120SQ-C—S) using the Biomek FX liquid handling system at 100× solutions of compounds in DMSO. 2×HPK1-MBP mix (final concentration 0.64 ng/μl of HPK1 and 45 ng/μl of MBP) was prepared in 1× Assay buffer and 5.5 μl of mixture per well was added into 384 w white Reaction plate with NBS (Corning, Cat #4513). 5.5 μl of MBP substrate w/o HPK1 in 1× buffer was used for negative control. Plates were centrifuged for 1 min at 100 g. Next step the Compounds were added to Reaction plate using Biomek station via following steps: 1l of 100× compounds (in DMSO) were mixed thoroughly with 49 ul of 2×10 uM ATP in Assay Buffer, then 5.5 μl of this mixture was added to Reaction plate with 5.5 μl of HPK1-MBP mix. Plates were centrifuged for 1 min at 100 g and incubated for 1 hour at room temperature. Next 3 μL of ADP-Glo reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added. Plates were incubated for 30 minutes at room temperature. Then 6 μL of Kinase detection reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added and the Luminescence was measured using Microplate Reader. |
| Affinity data for this assay | |
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