Monday, October 27, 2003
Description This protocol describes how to assay for kinase activity within a polyacrylamide gel, rather than in solution. The advantages to an in-gel assay are that an apparent molecular weight can be assigned to the kinase activity and that multiple kinase activities can be distinguished. In this assay, a polyacrylamide gel is cast with myelin basic protein, which acts as the substrate for the kinase reaction. After the samples containing the putative kinase(s) are electrophoresed on the gel, the kinase activity is assayed by soaking the gel in radiolabeled ATP. The kinase(s) will transfer the radiolabeled phosphate to the myelin basic protein at the position where the kinase resides in the gel. The molecular weight of the resulting band(s) on autoradiography indicates the apparent molecular weight of the kinase(s). A control polyacrylamide gel impregnated with BSA is also assayed. Any band that appears in this gel is due to autophosphorylation of the kinase itself. Procedure 1. Pour a 15% Polyacrylamide gel containing 0.5 mg/ml Myelin Basic Protein. Pour another 15% Polyacrylamide gel containing 0.5 mg/ml BSA as a control for bands arising on the gel due to autokinase activity. 2. Mix experimental samples containing the putative kinase activity with 1X Sample Buffer. Boil for 2 min. 3. Load samples and pre-stained molecular weight markers on gels (see Hint #1). 4. Electrophorese the gels until the dye front has run off the gel. 5. Wash the gels in Wash Buffer 1 for 1 hr at room temperature. Change the buffer 3 times during the wash incubation, using 500 ml each time. 6. Wash the gels in Wash Buffer 2 for 1 hr at room temperature. Use at least 10 times the original gel volume. 7. Wash the gels with gentle rocking in Wash Buffer 3 at 4C overnight, or for at least 16 hr. Change the buffer several times throughout the wash incubation. 8. Wash the gels in Kinase Buffer for 30 min at room temperature. Pour off the buffer. 9. Add 10 to 15 ml of Kinase Buffer containing 20 M ATP and 10 Ci/ml -[32P]-ATP to the gels (CAUTION! see Hint #2). 10. Incubate with rocking for 60 to 90 min at room temperature. 11. Wash the gels extensively with several changes of Wash Buffer 4 overnight, if possible, at room temperature. 12. Dry the gels on a gel dryer. 13. Detect kinase activity by autoradiography. Recipes Wash Buffer 4 5% (w/v) Trichloroacetic Acid 1% (w/v) Sodium Pyrophosphate Kinase Buffer 40 mM HEPES, pH 7.6 2 mM DTT 0.1 mM EGTA 10 mM Magnesium Acetate Wash Buffer 3 0.04% (v/v) Tween 20 50 mM Tris-Cl, pH 8.0 5 mM 2-Mercaptoethanol Wash Buffer 2 6 M Guanidine-HCl 50 mM Tris-Cl, pH 8.0 5 mM 2-Mercaptoethanol Wash Buffer 1 50 mM Tris-Cl, pH 8.0 20% (v/v) 2-Propanol Sample Buffer (2X) 2 mM DTT Add DTT just before use 20% (v/v) Glycerol 100 mM Tris-Cl, pH 6.8 0.2% (w/v) Bromophenol Blue 4% (w/v) SDS Supplies Tips 1. It is necessary to use prestained molecular weight markers. Because the polyacrylamide gel is impregnated with protein, it is not possible to stain the gel after the electrophoresis run to visualize the markers. 2. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions. (责任编辑:泉水) |