IN VIVO PHOSPHORYLATION/IP

A) GROWING AND LABELLING CELLS.

1. Grow cells of interest to 80% confluence in 150 mm dishes. Serum starve overnight in media containing 0.5% BSA (insulin-free), no serum.

2. Take cells to the hot room. Discard lids from plates and remove media, wash once with phosphate-free media, move cells into the hot box and add 5 ml of phosphate-free media containing 1-10 mCi ³²P-orthophosphate (NEN cat # NEX 053) per plate. Place cells in a lucite box and place in incubator 37^oC, 5% CO_{2 in hot room on rocker for 2-3 hrs to label to equilibrium.}

_{3. Stimulate cells for the desired time with the appropriate concentration of the desired growth factor (e.g. 1 min with 50 ul of 10}^-5 M insulin (for 10^-7 M final)).

4. Move cells to the hot box. Aspirate media quickly (hot waste), freeze cells by dousing in liquid nitrogen. Pour off excess LN2, add 2 ml/plate of solubilization buffer:

50 ml:

50 mM HEPES pH 7.4

1.0% Triton X-100 500 ul

2 mM Vanadate 18 mg

2 mM PMSF 500 ul of stock (35 mg/ml EtOH)

NaF 210 mg

5 mM EDTA 500 ul of 0.5 M stock

5. Thaw cells into buffer, scrape with cell lifter, transfer to polycarbonate centrifuge tubes with caps and spin in Ti70 rotor 55 krpm for 1 hour.

6. Immunoprecipitate supernatant with (20ul) of desired antibody (2 hr-overnight), collect on 100 ul pansorbin 1+ hours. Re-immunoprecipitate if desired.

7. Collect pellets-- spin down, aspirate supernatant (hot waste). Add 1 ml Tris-SDS IP wash, sonicate. Repeat 2 additional times. Aspirate supernatant, add 70 ul of laemmli sample buffer.

8. Boil 5', vortex to resuspend pellet, boil 3'. Spin down pansorbin 5', load supernatant to gel.