Protocol HotSpot Kinase Assay

The Assay

The HotSpot^TM Kinase Assay is used at our US facility for compound screening on protein kinases.

Reagents

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.

Procedure Step-by-Step

1. Prepare substrate in freshly prepared base reaction buffer
2. Deliver any required cofactors to the substrate solution above
3. Deliver indicated kinase into the substrate solution and gently mix
4. Deliver compounds in 100% DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature
5. Deliver ³³P-ATP into the reaction mixture to initiate the reaction.
6. Incubate kinase reaction for 2 hours at room temperature
7. Detect kinase activity by P81 filter-binding method.

Reference

Anastassiadis T, et al. Comprehensive assay of kinase catalytic activity reveals featuresof kinase inhibitor selectivity. Nat Biotechnol. 2011 Oct 30;29(11):1039-45. doi:
10.1038/nbt.2017.