1. Freeze Xenopus eggs in Liquid Nitrogen. Store at -80°C until needed.
2. Thaw oocytes by homogenizing in 20 μl of H1 Kinase Buffer per oocyte.
3. Centrifuge the homogenate for 3 min at maximum speed in a microcentrifuge at 4°C.
4. Take 9 μl of the supernatant (see Tip #1) and add 1 μl of H1 Kinase Buffer containing 1.25 μg Histone H1 (1.25 u*l of Histone H1 Solution also see Tip #2) and 0.63 μCi γ-[32P]-ATP (CAUTION! see Tip #3).
5. Incubate for 10 min at 30°C.
6. Stop the reaction by adding 30 μl of 2X Sample Buffer.
7. Load 10 μl 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 μl samples of Xenopus Egg extracts in Liquid Nitrogen. Store at -80°C until needed.
2. Make up enough H1 Kinase Reaction Mix for each reaction to contain (see Tip #4): 1X Reaction Buffer 1 mM DTT 200 μM ATP 1.25 μg Histone H1 (1.25 μl of Histone H1 Solution also see Tip #2) 0.63 μCi γ-[32P]-ATP (CAUTION! see Tip #3) Bring up the reaction volume to 10 μl with ddH2O.
3. Thaw the extract by adding 9 μl of the H1 Kinase Reaction Mix to each sample.
4. Incubate for 10 min at 30°C.
5. Stop the incubation by adding 10 μl of 2X Sample Buffer.
6. Load 8 μl 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 -80°C until needed.
4. Thaw cells by homogenizing in 20 μl of H1 Kinase Buffer.
5. Centrifuge the homogenate for 3 min at maximum speed in a microcentrifuge at 4°C.
6. Take 9 μl of the supernatant and add to 1 μl of H1 Kinase Buffer containing 1.25 μg Histone H1 (1.25 μl of Histone H1 Solution also see Tip #2) and 0.63 uCi γ-[32P]-ATP (CAUTION! see Tip #3).
7. Incubate the reaction for 10 min at 30°C.
8. Stop the incubation by adding 10 μl of 2X Sample Buffer.
9. Load 10 μl of the sample on a 15% Polyacrylamide gel (see protocol on SDS-PAGE).
10. Run the gel and process it for autoradiography (see protocol on Autoradiography).
Solutions and Buffers
Reaction Buffer (5X) 75 mM MgCl2 100 mM EGTA 400 μM Glycerol 2-Phosphate, pH 7.4
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 Tip #2)
H1 Kinase Buffer 1 mM DTT 10 μg/ml Soybean Trypsin Inhibitor (CAUTION! see Tip #3) 15 mM MgCl2 15 μg/ml Benzamidine 10 μg/ml Aprotinin (CAUTION! see Tip #3) 10 μg/ml Leupeptin (CAUTION! see Tip #3) 0.1% (v/v) Igepal CA630 20 mM EGTA 200 μM ATP 80 mM Glycerol 2-Phosphate, pH 7.4
BioReagents and Chemicals:
Nitrogen, Liquid DTT Glycerol Leupeptin ATP Soybean Trypsin Inhibitor Benzamidine IGEPAL CA-630 I3-[32P]-ATP Tris Bromophenol Blue EGTA Histone H1 SDS Aprotinin Magnesium Chloride Glycerol 2-Phosphate
Protocol 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.
