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 program

Materials and Instruments

Enzyme sample with cyclic nucleotide-dependent protein kinase activity 10 mg/ml Histone 2B (soluble in water)
[γ-32P] ATP solution 5 × cyclic nucleotide-dependent protein kinase reaction buffer 20 × cyclic nucleotide
30°C water bath

Move

1. For each analytical reaction, add the reaction components to a 1.5 ml microcentrifuge tube in the following proportions and maintain an ice bath:

5 × Cyclic Nucleotide-Dependent Protein Kinase Reaction Buffer

1 μl 10 mg/ml Histone 2B

1 [ ^γ-32P ] ATP solution (ATP final concentration 5 μmol/L, radioactivity 5 μlCi/μl)

1 μl 20 × cyclic nucleotide

0-13 μl water

Cover the tube, mix well, and place in a 30°C water bath.

Make three copies of each reaction and set up an experimental control without substrate, enzyme, cyclic nucleotide and other components. The volume of each reaction is 20 μl. 2.

2. Add 1-14 μl of pre-cooled enzyme sample with cyclic nucleotide-dependent protein kinase activity to start the reaction. The amount of enzyme added depends on the amount of enzyme activity in the sample, and the maximum reaction value obtained in the pre-test is a measure of the extent of the phosphotransfer reaction.

3. incubate at 30°C for 10 min.

4. Stop the reaction by adding the appropriate reagents for the different analytical methods: 20 μl of 10 % pre-cooled TCA for TCA precipitation or 10-20 μl of pre-cooled 2 × SDS-PAGE sample buffer for electrophoretic analysis.

Caveat

1. The amount of enzyme added in the reaction can be reduced in order to maintain the linear binding relationship between the phosphate group and the substrate.

2. For immunoprecipitated enzyme samples adsorbed on Sepharose beads, prepare a reaction mixture without enzyme source, preheat and add the immunoprecipitate. For immunoprecipitate analysis, it is recommended to expand the volume to 100 μl by adding 25 μl of gel beads adsorbed with immunoprecipitate to 75 μl of reaction buffer.

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