Lysis, IP, Western

1. Cut approximately 50 sections of frozen tissue (30um each).

2. Add 200l lysis buffer + 1:50 PMSF + 1:500 aprotinin; boil 5 min.

3. Sonicate 2 x 10 sec. (setting 6) with 10 sec. interval on ice.

4. Boil 5 min. Spin 15 min., 14,000 rpm. Take supn and discard pellet

5. Dilute 10l of each sample in 40l TE; perform protein assay.

6. Equalize all protein amounts to 200-500ug. Make all volumes up to 200l with lysis buffer + 1:50 PMSF + 1:500 aprotinin.

7. Wash beads with an equal volume of 1X PBS (spin 2 min., 14,000 rpm). Resuspend beads 1:1 in 1X PBS.

8. Add 1mL Solution A + 1:50 PMSF + 1:500 aprotinin to each tube of protein/lysis buffer. Spin 15 min., 14,000 rpm.

9. Add supernatant from #8 to new labeled eppendorfs containing:

30l beads

5l rabbit anti-mouse antibody

10l 9E10 antibody

10. Rotate tubes overnight at 4 C; spin down beads at 4 C (2 min., 14,000 rpm).

11. Wash beads 6X with 1mL wash buffer + 1:50 PMSF + 1:500 aprotinin (each wash is a 2- minute spin at 4 C, 14,000 rpm).

12. Add 100l 1X Laemmli buffer to each tube of beads; keep on ice (or freeze) until ready to boil.

13. Boil beads + Laemmli buffer 7 min. Spin samples briefly. Load 50l of each sample (+ 20l Gibco marker) on Laemmli gel (use 7.5% running gel and 3.8% stacking gel); run at 50-60mA.

14. Transfer to nitrocellulose membrane at 350-440 mA in 1X Transfer Buffer.

15. Stain 5 min. in Ponceau; rinse with 1X TBS, 0.1% Tween.

16. Block membrane with 5% milk, 1X TBS, 0.1% Tween 1 hour at room temp.

17. Incubate in primary antibody (9E10) diluted 1:4000 in 5% milk, 1X TBS, 0.1% Tween 1-2 hr. at room temp. or O/N at 4 C; wash 3 x 10 min. in 1X TBS, 0.1% Tween.

18. Add secondary antibody (HRP) diluted 1:4000 in 5% milk, 1X TBS, 0.1% Tween. Incubate 90 min. at room temp.; wash 3 x 10 min. in 1X TBS, 0.1% Tween.

19. Prepare ECL reagent (0.125ml/cm2 membrane). In darkroom, pour reagent over membrane; let it sit 1 min. without agitating. Blot off liquid on Whatman paper, place membrane inside plastic wrap, and immediately expose to film inside cartridge. Develop film after increasing intervals, starting with 30 seconds.