|
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.
|
