1. Place dishes with transfected cells on ice (metal plate on ice-bucket on a rocker) and wash three times with ice-cold PBS.
2. Add ice-cold Lysisbuffer (+pi=mammalian protease inhibitors @ 1:250) to the dish. Use 0.5 ml for 6 cm dish, 1 ml for 10 cm dish and 2.5 ml for 15 cm dish. Keep rocking on ice for 15 min (make sure buffer covers the whole surface). Swell proteinA-sepharose in millipore water in a 15 ml conical (mix well to resuspend all the beads).
3. Harvest the supernatant into 1.5 or 2 ml eppendorf tubes. Pellet any insoluble material for 3 min at 9000 g at 4°C. Collect the supernatant and discard the pellet.
4. Protein assay the sample(s). Bring the lysate to 1 mg/ml protein concentration with lysisbuffer/pi. Save 10-100 µg of lysate for gels. Wash the proteinA-sepharose twice with water and add 1 volume of millipore or buffer to one bed-volume (use 2 min at 1000 rpm for pelleting.
5. Add 3-5 µl of rabbit polyclonal anti-HA antiserum (Babco: aliquots in —20°C) per mg total protein (change tips between pipetting ). Then add 25 µl ProteinA-sepharose (50% slurry in water/buffer) per mg protein with a cut-off yellow tip (flick well to keep beads in suspension between pipetting) .
6. Incubate on wheel in cold-room 2 -4 hours.
7. Pellet beads for 30 seconds in eppendorf centrifuges at top speed or 2 min in large centrifuge at 1000 rpm. Save unbound.
8. Wash beads 3x with YP-washbuffer/pi. Pellet as above and remove all but 10-20 µl of wash to prevent loosing beads.
9. If the IP is to be loaded on an SDS-PAGE, wash 1x with 10 mM Tris pH 7.5 to remove detergent, leave ~20 µl buffer on the beads and add 10 µl of 5x sample buffer. If the IP is to be used for other purposes use adequate buffer for the final wash.

25 mM Tris pH 7.5 5 mls 0.5 M

Only for Y-Phasphorylation experments:

Add 50 mM NaF (0.21 g) and 1/100 Phosphatase Inhibitor cocktail II