Experiments for direct analysis of kinases in gels
Experiments for direct analysis of kinases in gels
Protein kinases are analyzed using labeled donor substrates, and the accumulation of markers in protein or peptide acceptor substrates is readily detected when the enzyme sample contains phosphotransferase activity. From The Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
Basic scenarios Direct analysis
Principle
Guanidine hydrochloride or urea both denature proteins; which one is chosen depends on the different kinases, but guanidine hydrochloride is generally preferred because it denatures better for most kinases.
Materials and Instruments
10 mmol L kinase substrate Enzyme sample with kinase activity Move 1. prepare a polyacrylamide separator gel at the appropriate percentage concentration by adding 10 mg/ml of kinase substrate to achieve a final concentration of 1 mg/ml in the separator gel mixture. mix homogeneously and pour into the gel device to polymerize. 2. Prepare SDS-PAGE electrophoresis samples of the kinase-activated samples in the usual way and electrophorese. 3. 3. After electrophoresis, put the gel into 200 ml of 20 % (V/V) 2-propanol/50 mmol/L Tris-Cl, pH 8.0 wash solution, rinse for 20 min, replace with new buffer, and repeat twice. 4. 4. rinse three times for 20 min with 200 ml of 1 mmol/L DTT/50 mmol/L Tris-Cl pH 8.0. 5. rinse three times for 20 min with 250 ml of 6 mmol/L DTT/50 mmol/L Tris-Cl pH 8.0. 5. wash twice with 250 ml of 6 mol/L guanidine hydrochloride/1 mmol/L DTT/50 mmol/L Tris-Cl pH 8.0 or 8 mol/L urea/1 mmol/L DTT/50 mmol/L Tris-Cl pH 8.0 for 30 min. 6. Wash the gel 8-10 times with 250 ml of 1 mmol/L DTT/0.05 % (V/V) Tween 20/50 mmol/L Tris-Cl pH 8.0 at 4°C during the cycle of 18 h or more to fully revert the proteins. 7. Place the gel into 250 ml of kinase-matched reaction buffer (containing 10 mmol/L MgCl2 ) at 30°C in a warm bath for 20 min. 8. Remove any residual buffer. 8. Remove all residual buffer and place the gel into a container with a tight-fitting lid or a heat-sealable polyethylene bag (containers should be specific for radioactive materials). Add as little Kinase Reaction Buffer as necessary to cover the gel surface. Add 10 mmol/L Mg/ATP solution (final concentration 50 μmol/L ATP) in 1/4 the volume of the reaction buffer, and finally add 20 μCi/ml [ γ-32P ] ATP. 9. 9. 30°C, warm bath for 1 h. 10. 10. Soak the gel in 250 ml of 5 % TCA for 15 min and repeat once. Wash the gel in 500 ml of 1 % sodium pyrophosphate/5 % TCA and repeat the wash until the wash solution is almost free of radioactive material. 11. The gel is dried on filter paper and radiographically autoradiographed. Caveat Because of the radioactivity of the samples, extra care should be taken during sample preparation and transfer. Relevant reactions and manipulations should be carried out in microcentrifuge tubes with screw caps to minimize32P radioactive contamination hazard. In electrophoresis, the dye must be transferred out of the gel before proceeding to the next step, as this allows residual [γ-32P] ATP is transferred out of the gel along with the dye, which also means that the residual [γ- 32 P] ATP in the electrophoresis buffer will be removed from the gel.32P in the electrophoresis buffer, which should be disposed of according to the principles of safe disposal of radioactive waste liquids. Common Problems Materials: 10 mmol/L kinase substrate such as myelin basic protein Enzyme samples with kinase activity (see cell lysis assay for kinase analysis), maintained in an ice bath Reagents: 20% (V/V) 2-propanol / 50 mmol/L Tris - Cl (pH 8.0 at room temperature) 1 mmol/L DTT / 50 mmol/L Tris - Cl (pH 8.0 at room temperature) 6 mol/L guanidine hydrochloride / 1 mmol/L DTT / 50 mmol/L Tris - Cl (pH 8.0 at room temperature) or 8 mol/L urea / 1 mmol/L DTT / 50 mmol/L Tris - Cl (pH 8.0 at room temperature) 1 mmol/L DTT/0. 05% (V/V) Tween 20 /50 mmol/L Tris - Cl (pH 8.0 at 4°C) Matching kinase reaction buffer 10 mmol/L Mg/ATP solution 10 mCi/ml [ γ-32P ] ATP (3000 Ci/mmol; Amersham, DuPont NEN or ICN Biomedicals) 5 % (m/V) trichloroacetic acid (TCA) 1 % (m/V) sodium pyrophosphate /5% (m/V) TCA For more product details, please visit Aladdin Scientific website.
2-propanol Tris-Cl guanidine hydrochloride urea DTT Tween 20 matching kinase reaction buffer Mg/ATP solution [γ-32P] ATP trichloroacetic acid (TCA) sodium pyrophosphate
Specialized warm baths for radioactive substances (e.g., small tray with tight lid and heat-sealable polyethylene bag sealer (optional))