| Assay Method Information | |
| | HPK1 Kinase Activity Inhibition Assay |
| Description: | 1. HPK1 Kinase Activity Inhibition Test. The kinase activity of HPK1 is manifested as activity of autophosphorylation and phosphorylation of downstream substrates. In the process of autophosphorylation, additional substrates are not required, and ATP is consumed to generate ADP. The amount of the product was measured by ADP-Glo reagent and luminescence method to reflect kinase activity.Test compounds: compounds prepared in the examples of this application.Prepare compound stock solution: dissolve the compound to be tested in 100% DMSO to make a 10 mM stock solution;Prepare 4× Kinase Reaction Buffer:Concentration of FinalMaterial Stock Solution Volume ConcentrationTris 1M (25×) 240 μL 40 mMMgCl2 1M (50×) 120 μL 20 mMBSA 7.5%(75×) 80 μL 0.1%DTT 1M(500×) 3 μL 0.5 mM ddH2O 5557 μLPrepare 2×HPK1 Kinase Solution:Concen- tration 2 × Final Finalof Stock Concen- Concen-Material Solution Volume tration trationHPK1 4878 nM 1 μL 10 nM 5 nM1 × kinase 487 μLreaction buffersolutionPrepare 4×ATP mixture:Concen- 4 × tration Final Finalof Stock Concen- Concen-Material ATP Km Solution Volume tration trationATP 1.669 μM 1 mM (125×) 3 μL 8 μM 2 μM4 × kinase 372 μL reaction buffersolution Procedures:Dilute the stock solution of the compound to be tested by 5 times with 1000 DMSO, make a 4-fold equal dilution in a 96-well dilution plate, add 1 μL of the compound to 49 L of kinase reaction buffer, and shake on a microplate shaker for 20 minutes. Transfer 2 μL of 2×HIPK1 kinase solution to 384 reaction plate, add 1 μL of the test compound to the 384 reaction plate (Greiner, 784075), centrifuge for 1 minute (1000 rpm/min), incubate at 25° C. for 10 minutes. Transfer 1 L of the 4×ATP mixture to a 384 reaction plate, centrifuge for 1 minute (1000 rpm/mm), and incubate at 25° C. for 60 minutes. In the reaction system, the final concentration of DMSO was 0.500. Transfer 4 μL of ADP-Glo to a 384 reaction plate, centrifuge for 1 minute (1000 rpm/min), and incubate at 25° C. for 40 minutes. Transfer 8 μL detection solution to a 384 reaction plate, centrifuge for 1 minute (1000 rpm/min), and incubate at 25° C. for 40 minutes. The fluorescence signal was read using a Biotek multi-function plate reader, and the +C50 (half inhibitory concentration) of the compound was obtained using a four-coefficient nonlinear fitting formula. |
| Affinity data for this assay | |
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