Immunopurification of Tyrosine Phosphorylated Proteins

Cell Lysis

Non-Denaturing Conditions

1. Rinse adherent or non-adherent cells from a 15 cm dish with an excess of phosphate-buffered saline (PBS); repeat wash.
2. Lyse cells with 6 ml of ice-cold lysis buffer (10 mM imidazole pH 7.3, 0.5 M NaCl, 1% Triton X-100, 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 2 mM sodium azide) with gentle rotation for 30 minutes at 4°C.
3. Remove the lysate from the plate and centrifuge at 40,000 rpm for 1.5 hour at 4°C.

Denaturing Conditions

1. Rinse adherent or non-adherent cells from a 15 cm dish with an excess of phosphate-buffered saline (PBS); repeat wash.
2. Lyse cells with 3 ml of boiling lysis buffer (1% SDS, 10 mM Tris pH 7.4, 0.2 mM sodium ortho-vanadate).
3. Microwave the cells for 5 seconds to assure complete lysis and denaturation.
4. Scrape the lysate from plates and centrifuge at 40,000 rpm for 1.5 hour at 15°C.
5. Dilute the lysate 5-fold to a final concentration of 0.2% SDS, adjusted to 10 mM imidazole pH 7.3, 50 mM NaCl, 1% Triton X-100, 0.2 mM sodium ortho-vanadate, 1 mM EDTA.

Purification

1. Equilibrate PY20 coupled Affi-Gel 10 (Bio-Rad) in cold resin wash buffer (10 mM imidazole pH 7.3, 50 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA) by repeated centrifugation and resuspension in resin wash buffer.
2. Incubate the lysate (up to 250 mg total protein) with 3 ml of anti-phosphotyrosine PY20 coupled to Affi-Gel 10 overnight at 4°C with constant rotation.
3. Collect the Affi-Gel by low speed centrifugation, resuspend in resin wash buffer, and load into a small column.
4. Wash the column with 20 volumes of resin wash buffer.
5. Wash the column with three volumes of low-detergent resin wash buffer (10 mM imidazole pH 7.3, 50 mM NaCl, 0.1% Triton X-100, 0.05% SDS, 1 mM EDTA).
6. Elute the tyrosine-phosphorylated proteins with 5 mM phenyl phosphate in the low-detergent buffer. The eluted proteins can be monitored by protein determination or Western blotting with anti-phosphotyrosine antibodies.
7. The column should be regenerated by extensive washing with a neutral pH buffer (like PBS) containing 1 M NaCl, followed by several washes with PBS alone. Store the column at 4°C in PBS + 1.5 mM sodium azide between uses.