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. Content from Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
basic program
Materials and Instruments
Rabbit muscle enolase Move 1. Centrifuge the sample equivalent to 100 μg of Rabbit Muscle Enolase at maximum speed for 5 min at 4℃ and discard the supernatant. 2. 2. Add 10 μl of 1 mmol/L DTT/50 mmol/L HEPES, pH 7.0, mix thoroughly, and ice bath for 30~60 min. 3. 3. Add 10 ml glycerol and mix well. If subsequent experiments are performed on the same day, keep in an ice bath, otherwise store at -70℃. 4. 4. Add 20 μl of 100 mmol/L acetic acid and mix well before the experiment. 30°C water bath for 5 min. keep in an ice bath until the next step. 5. 5. For each analytical reaction, add the reaction components to a 1.5 ml microcentrifuge tube in the following proportion: 4 μl 5 × tyrosine kinase reaction buffer 1 μl acid-denatured enolase (2.5 μg enolase) 1 μl [ γ-32P ] ATP solution (ATP final concentration 5 μmol/L, radioactivity 5 μCi/μl) 0 to 14 μl water Cover centrifuge tubes and water bath at 30°C for 10 min. The amount of water added depends on the amount of enzyme sample added. The volume of water to be added depends on the amount of enzyme sample added. 6. Start the reaction by adding 1 to 14 μl of the enzyme sample with tyrosine kinase activity. 7. 7. Incubate in a water bath at 30°C for 10 min. 8. 8. Stop the reaction by adding 20 μl of pre-cooled 2 × SDS-PAGE sample buffer, mix thoroughly, and incubate in a boiling water bath for 3 min. 20 μl of the buffer should be analyzed by SDS-PAGE electrophoresis. Caveat 1. Do not leave the acid-denatured enzyme in the ice bath for more than 1 h. 2. The volume of enzyme added in step 6 depends on the amount of enzyme activity in the sample. The maximum reaction value obtained in the pre-test provides a measure of the extent of the phosphate transfer reaction. The volume of enzyme added during the reaction can be reduced in order to maintain a linear binding relationship between the phosphate group and the substrate. For more product details, please visit Aladdin Scientific website.
1 mmol L DTT 50 mmol L HEPES (pH 7.0) Glycerol 100 mmol L Acetic acid 5 × Tyrosine kinase reaction buffer [γ-32P] ATP solution Ice 2 × SDS-PAGE sample buffer
30°C Water bath and boiling water bath