Protocol/In Vitro Kinase Assay

Protocol for In Vitro Kinase Assay

Materials and Reagents

Immunoprecipitation Buffer

• 10 mM Tris, pH 7.4
• 1.0% Triton X-100
• 0.5% Nonidet P-40
• 150 mM NaCl
• 20 mM Sodium fluoride
• 0.5 mM Sodium orthovanadate
• 1 mM EDTA
• 1 mM EGTA
• 10 mM Sodium pyrophosphate
• 0.2 mM PMSF
• 10 μg/mL Aprotinin
• 10 μg/mL Leupeptin
• 10 μg/mL Pepstatin A

Kinase Buffer

• 10 mM Tris, pH 7.4
• 150 mM NaCl
• 10 mM MgCl2
• 0.5 mM Dithiothreitol (DTT)

Staining Solution

• 0.25% Coomassie blue
• 45% Methanol
• 10% Acetic acid

Destaining Solution

• 40% Methanol
• 10% Acetic acid 5× Electrophoresis Buffer
• 625 mM Tris, pH 6.8
• 20% SDS
• 50% Glycerol
• 0.05% Bromophenol blue
• 10% β-Mercaptoethanol
• 0.1 M Sodium orthovanadate (phosphatase inhibitor). Dissolve powder in water, pH 10.0. Boil. Keep at room temperature for up to 1 week in the dark.
• 10 mg/mL of aprotinin and leupeptin in water. Keep frozen at -20°C in aliquots.
• 10 mg/mLof pepstatin A in ethanol. Keep at -20°C.
• [γ-32P]ATP
• Cold ATP
• X-ray film (Amersham Pharmacia

Biotech)

Procedure

Preparation of Cell Lysate

1. Wash cells on a confluent 100-mm culture dish with 10 mL of PBS.

2. Lyse the cells by addition of 1 mL cold immunoprecipitation buffer.

3. Scrape the cells off the dish and pass 5 to 10 times through a 26 gauge needle to disperse large aggregates, then incubate for 20 minutes on ice.

4. Centrifuge (14 000× g) for 30 minutes in a microcentrifuge at 14 k rpm at 4°C. Retain the supernatant.

5. Repeat centrifugation in a new tube.

6. The supernatant is the total cell lysate. Measure the protein concentration using the BCA kit and protein standards. Immunoprecipitation of the Protein Kinase

7. Incubate the cell lysate (0.5–1.0 mg protein) with 2 to 5 μg soluble antibody.

8. Immunoprecipitate for 1 hour at 4°C on a tube turner.

9. Add 30 μL of 50% protein A-sepharose suspension and incubate for 2 hours at 4°C on a tube turner.

10. Wash the complexes by resuspension in immunoprecipitation buffer, followed by a 3-minute centrifugation in a microcentrifuge 14 k rpm at 4°C. Repeat the wash twice.

11. Collect the complexes by centrifugation for 3 minutes in a microcentrifuge at 14 k rpm at 4°C. Kinase Assay

12. Wash the immunocomplexes three times at 4°C with kinase buffer.

13. Remove the supernatant by aspiration and, with the pellet on ice, add 40 μL of kinase buffer containing the appropriate protein substrate at 1.0 mg/mL (e.g., 5 μg of acid denatured enolase), 25 μM cold ATP, 2.5 μCi [γ-32P]ATP.

14. Mix carefully by pipetting up and down.

15. Transfer the tubes to 30°C in a circulating water bath and incubate for 15 minutes.

16. Add 15 μL of boiling 5× concentrated electrophoresis sample buffer to stop the reaction. Boil for 5 minutes.

17. Centrifuge the samples and electrophorese the soluble fractions.

18. Fix and stain the gel in staining solution for 45 minutes at room temperature.

19. Destain the gel in destaining solution for 2 hours. Change the destaining solution 4 to 5 times.

20. Dry the gel and expose it to X-ray film at -80°C. Kinase activity will be indicated by a band of phosphorylated protein substrate.

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