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Star Republic: Guide for Biologists

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.