HISTONE KINASE ASSAY

1. Histone kinase activity is frequently immunoprecipitated with antibody from cell extract. Cell extract can also be used directly.

2. Prepare the following reaction mix:

Histone kinase activity (e.g. CDK) bound on antibody/argarose-beads
Histone kinase buffer
(50 mM HEPES pH 7.5, 1 mM DTT, 10 mM MgCl2 , 2.5 mM EGTA, 0.1 M sodium vanadate, 10 mM b-glycerol phosphate, 1 mM NaF, 20 mM ATP)
20 ml
histone H1 (1-10 mg) 1 ml
[g-32P]ATP (10 mCi) 1 ml

3. Incubate at 30 or 37 C for 30 minutes.

4. Stop the reaction by adding SDS-PAGE loading buffer and analyze by SDS-PAGE.

4a. Alternatively, stop the reaction by adding equal volume of 10% TCA and measure 32P incoporation using cellulose phosphate filter and scintillation counting.