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 samples with PKC activity
[γ-32P] ATP Solution PKC Reaction Buffer TCA SDS - PAGE Sample Buffer
30°C Water Bath P81 Cellulose Phosphate Membrane Tube

Move

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

4 μl 5 × PKC reaction buffer

1 μl of 10 mg/ml Histone H1

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

0-14 μl water

Cover the centrifuge tube and incubate in a 30℃ water bath for 10 min.

Three reactions were set up as a group without substrate and enzyme control. The volume of each reaction is 20 μl. The amount of water added in step 2 depends on the amount of enzyme sample used. 2.

2. Start the reaction by adding 1-14 μl of PKC-active enzyme sample to the preheated reaction mixture. 3.

3. Incubate at 30°C for 10 min. 4.

4. Stop the reaction by adding the appropriate reagents for the different analytical methods: 20 μl of 10 % pre-cooled TCA for TCA precipitation, 10 μl or 20 μl of pre-cooled 2 × SDS-PAGE sample buffer for electrophoresis. Samples can be adsorbed onto a P81 cellulose phosphate membrane by spotting the membrane with 10 μl of reaction product. Continue analysis by one of these methods.

Common Problems

Enzyme samples with PKC activity (see Supporting Program 1)

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