Monday, October 27, 2003

Description
This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

Procedure
A. H1 Kinase Assay on Individual Xenopus Oocytes

1. Freeze Xenopus eggs in Liquid Nitrogen. Store at -80C until needed.

2. Thaw oocytes by homogenizing in 20 l of H1 Kinase Buffer per oocyte.

3. Centrifuge the homogenate for 3 min at maximum speed in a microcentrifuge at 4C.

4. Take 9 l of the supernatant (see Hint #1) and add 1 l of H1 Kinase Buffer containing 1.25 g Histone H1 (1.25 u*l of Histone H1 Solution also see Hint #2) and 0.63 Ci -[32P]-ATP (CAUTION! see Hint #3).

5. Incubate for 10 min at 30C.

6. Stop the reaction by adding 30 l of 2X Sample Buffer.

7. Load 10 l of the sample on a 15% Polyacrylamide gel (see Protocol on SDS-PAGE).

8. Electrophorese the gel and process it for autoradiography (see Protocol on Autoradiography).

B. H1 Kinase Assay on Xenopus Egg Extract Samples

1. Freeze 1 l samples of Xenopus Egg extracts in Liquid Nitrogen. Store at -80C until needed.

2. Make up enough H1 Kinase Reaction Mix for each reaction to contain (see Hint #4):

1X Reaction Buffer

1 mM DTT

200 M ATP

1.25 g Histone H1 (1.25 l of Histone H1 Solution also see Hint #2)

0.63 Ci -[32P]-ATP (CAUTION! see Hint #3)

Bring up the reaction volume to 10 l with ddH2O.

3. Thaw the extract by adding 9 l of the H1 Kinase Reaction Mix to each sample.

4. Incubate for 10 min at 30C.

5. Stop the incubation by adding 10 l of 2X Sample Buffer.

6. Load 8 l of the sample on a 15% Polyacrylamide gel (see Protocol on SDS-PAGE).

7. Run the gel and process it for autoradiography (see Protocol on Autoradiography).

C. H1 Kinase Assay on Tissue Culture Cells

1. Centrifuge the cells at 1,000 X g for 5 min in a table-top centrifuge or equivalent to pellet the cells.

2. Aspirate the supernatant.

3. Freeze the cells in Liquid Nitrogen. Store at -80C until needed.

4. Thaw cells by homogenizing in 20 l of H1 Kinase Buffer.

5. Centrifuge the homogenate for 3 min at maximum speed in a microcentrifuge at 4C.

6. Take 9 l of the supernatant and add to 1 l of H1 Kinase Buffer containing 1.25 g Histone H1 (1.25 l of Histone H1 Solution also see Hint #2) and 0.63 uCi -[32P]-ATP (CAUTION! see Hint #3).

7. Incubate the reaction for 10 min at 30C.

8. Stop the incubation by adding 10 l of 2X Sample Buffer.

9. Load 10 l of the sample on a 15% Polyacrylamide gel .

10. Run the gel and process it for autoradiography

Recipes
Sample Buffer (2X) Add DTT just before use.
2 mM DTT
20% (v/v) Glycerol
100 mM Tris-Cl, pH 6.8
0.2% (w/v) Bromophenol Blue
4% (w/v) SDS


Histone H1 Solution 1 mg/ml Histone H1 (Boehringer Mannheim; see Hint #2)


H1 Kinase Buffer 1 mM DTT
10 g/ml Soybean Trypsin Inhibitor (CAUTION! see Hint #3)
15 mM MgCl2
15 g/ml Benzamidine
10 g/ml Aprotinin (CAUTION! see Hint #3)
10 g/ml Leupeptin (CAUTION! see Hint #3)
0.1% (v/v) Igepal CA630
20 mM EGTA
200 M ATP
80 mM Glycerol 2-Phosphate, pH 7.4


Reaction Buffer (5X) 75 mM MgCl2
100 mM EGTA
400 M Glycerol 2-Phosphate, pH 7.4



Supplies


Tips
1. This is equivalent to half an oocyte.

2. The contributor of this protocol suggests that users not use H1 Kinase from Sigma.

3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

4. There is no need for detergent, benzamidine, or protease inhibitors for frog extracts.