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. (责任编辑:泉水) |