Histone H1 Kinase Activity Assay

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Histone H1 Kinase Activity Assay

Date Added: May 27, 2008 05:43:58 AM

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Category: Epigenetics and Methylation Protocols

Histone H1 Kinase Activity Assay Protocol

A. H1 Kinase Assay on Individual Xenopus Oocytes

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.

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