- g [32 P] ATP (0.5 - 1x106 c.p.m./nmol) 4mM ATP solution in water: dilute from a 100mM stock, adjust pH to 7.0 using NaOH; store at -20šC 20mg/ml Histone H1 solution in water; store at -20šC
- Extraction buffer (EB): 80mM b-glycerophosphate (pH 7.3), 20mM EGTA , 15mM MgCl2, store in aliquots at -20šC; just prior to use add 1mM DTT and protease inhibitors (1mM PMSF, 10µg/ml each of Aprotinin, Pepstatin and Leupeptin) in EB diluted sample.
- For mitotic extract we use 1:20 dilution 96 micro-well plate Phosphocellulose P81 paper (Wathman) 0-20µl pipette and tips forceps and scissors beaker with wash solution (150mM H3PO4) technical grade ethanol scintillation counter and vials
A: Kinase reaction :
Prepare the assay mix of g [32 P] ATP-histones at 4šC (sufficient for 60 assays): 3µl of g [32 P] ATP (0.5 - 1x106 c.p.m./nmol), 45µl of 4mM ATP, 222µl of EB, 30µl of 20mg/ml histone H1. Pipette 2 x 5µl of each sample to be assayed into labelled micro-well plate , including two blanks of EB at 4šC. Warm the micro-well plate to 20šC. Start each reaction by adding 5µl of each sample, mix well by pipetting up and down several times. Start and stop reaction at 15sec intervals. Let each reaction continue for 15min at 20šC. Stop reaction by transferring 6µl of the reaction mixture to phosphocellulose P81 paper precut into 1.5cm squares. Paper filters should be identified with pencil (no ink) so they can be washed in the same beaker. Allow mixture to adsorb for a few seconds then dip into a beaker containing 150mM H3PO4. Wash 3x15min in 150mM H3PO4 at 20šC. Rinse briefly in ethanol and air-dry on paper towels. Transfer strips into scintillation vials. Add scintilation fluid and count (c.p.m.*). Count two 5µl samples of the assay mix adsorbed onto phosphocellulose paper to determine the ATP specific radioactivity (c.p.m.**)
B: Calculations :
Average the c.p.m. values for the controls to get the background. Substract this from the c.p.m. for each sample. Substract the control value from the c.p.m. values for the 5µl mix and calculate the average. The ATP concentration in the assay is that due to the 5µl of the assay mix (3000pmol). When assaying extracts add the ATP concentration due to the 5µl diluted extract (250pmol, assuming 1mM ATP in undiluted extract). Kinase activity in pol/min/µl:
= c.p.m.* x (pmol ATP in assay / c.p.m.** in ATP) x (sample volume / counted volume) x (1 / volume original extract in assay) x (1 / assay time)
= c.p.m.* x (3000 / c.p.m.** in ATP) x (10µl / 6µl) x (1 / 0.25µl) x (1 / 15min)
= c.p.m.* x (1333 / c.p.m. in ATP)
c.p.m.* = counts in experimental samples.
c.p.m.** = total c.p.m. as determined above
Heike Wilhelm, Eric Karsenti
