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Important Remarks:
- Rinse all plastic vessels with BSA solution (10 mg/mL in lysis buffer). Use lipid-free BSA, e.g., Sigma A7906.
- For each immunoprecitation setup, pre-incubate 50 µL of well-suspended magnetic beads (10 mg/mL in lysis buffer) with 500 µL BSA solution at RT for 30 minutes.
- Pellet pre-incubated beads before use by magnetic force or centrifugation. Discard supernatant.
- Cell extracts contain a large number of phosphorylated proteins. Using antibodies directed against phospho amino acids, the precipitate will contain a variety of different phosphoproteins. In contrast to immunoprecipitation of single proteins, where less primary antibody is required, we recommend using 10 µg phospho amino acid specific primary antibody for immunoprecipitation of phosphoproteins from lysates of 106 cells.
Preparation of Cell Extracts
Lysis Buffer
20 mM Imidazole-HCl, pH 6.8
100 mM KCl
1 mM MgCl2
10 mM EGTA
0.2% (v/v) Triton X-100
10 mM NaF
1 mM Sodium Vanadate
1 mM Sodium Molybdate
10 mM Sodium Pyrophosphate
25 mM ß-Glycerophosphate
Aliquote and store frozen!
Procedure
- Suspend cells in cell lysis buffer (1 mL buffer per 107 cells).
- Incubate at room temperature for 5 minutes, or until cell lysis is terminated.
- Centrifuge at 14,000 x g, 4°C for 5 minutes. Use supernatant for immunoprecipitation. It is important to use freshly prepared supernatants for IP, since otherwise dephosphorylation may occur.