Protocol HotSpot Kinase Assay
The HotSpotTM Kinase Assay is used at our US facility for compound screening on protein kinases.
Base reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO
Required cofactors are added individually to each kinase reaction.
- Prepare substrate in freshly prepared base reaction buffer
- Deliver any required cofactors to the substrate solution above
- Deliver indicated kinase into the substrate solution and gently mix
- Deliver compounds in 100% DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature
- Deliver 33P-ATP into the reaction mixture to initiate the reaction.
- Incubate kinase reaction for 2 hours at room temperature
- Detect kinase activity by P81 filter-binding method.
Anastassiadis T, et al. Comprehensive assay of kinase catalytic activity reveals features
of kinase inhibitor selectivity. Nat Biotechnol. 2011 Oct 30;29(11):1039-45. doi: