Assay Protocol HotSpot
Assay Protocol HotSpot

Protocol HotSpot Kinase Assay

The 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.

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.


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: