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)
|histone H1 (1-10 mg)
|[g-32P]ATP (10 mCi)
3. Incubate at 30 or 37 °C for 30 minutes.
4. Stop the reaction by adding SDS-PAGE loading buffer and analyze by
4a. Alternatively, stop the reaction by adding equal volume of 10% TCA and
measure 32P incoporation using
cellulose phosphate filter and scintillation counting.