Protocol for Western Blotting of Tissue Culture Cells

1. Grow cells to confluency
2. Remove media and wash with PBS
3. Remove PBS and add aprporiate volume of a freshly made solution of 0.15 units/ml aprotinin, 1mM PMSF, and 1 mM sodium orthovandadate (~200ul per 100mm plate). This may require a larger volume and serial transfer to successive plates
4. Incubate at 4^oC rotator for ~1 hour
5. Boil sample for three minutes and pass through 21 gauge needle and 26 gauge needle
6. Add an additional 5ul of 200mM PMSF
7. Centrifuge at 10,000rpm for 15minutes
8. Discard pellet
9. Supernatant may be stored at -70^oC until analysis. If frozen, samples should be boiled for ~2 minutes prior to electrophoresis
10. Load protein gel with 10-20ul of lysate per well (depends on gel thickness)
11. Transfer onto nitrocellulose membrane using electroblotting
12. Block membrane for 1 hour at room temperature or overnight at 4^oC in TBS containing 5% non-fat powdered milk
13. Wash membrane twice briefly (10-15 minutes) in TBS containing 0.05% Tween-20
14. Incubate nitrocellulose membrane with primary antibody solution (~0.5ug/ml - requires optimization) in TBS with 0.3% tween-20 and 0.1% BSA for 60 minutes at room temperature
15. Wash membrane twice briefly (10-15 minutes) in TBS containing 0.05% Tween-20
16. Incubate nitrocellulose membrane with HRP conjugated secondary antibody (~0.5ug/ml - requires optimization) in TBS with 0.3% tween-20 and 0.1% BSA for 60 minutes at room temperature
17. Wash nitrocellulose membrane by soaking in TBS with 0.05% tween for ten minutes. Repeat twice for a total of three washes
18. Wash nitrocellulose membrane twice with TBS without tween-20
19. Remove nitrocellulose membrane and blot dry
20. Develop via a number of commercially available detection kits