Silver: In Vivo Protein Methylation Assay (S. cerevisiae)

Uses ³H-Met from Amersham-Pharmaracia (5 mCi/ml)

• Notes:
  • Complete SD Met- media with cyclohexamide/chloramphenicol as below, need this for later washes/resuspension.
  • Working with ³H-Met – make sure you do the swipe test after experiments.
  • Make 2 SDS-PAGE gels during the 2h incubation stage.

• Protocol

1. Grow cells to early log phase, need about 20 ml.
2. Inhibit protein synthesis by the addition of cyclohexamide (1000x = 100 mg/ml)& chloramphenicol (1000x = 40 mg/ml), final conc = 100µg/ml for cyclohexamide & 40 µg/ml for chloramphenicol.
   • Make cyclohexamide in ethanol; make chloramphenicol in ethanol (kept in dark with foil, 4°C)
     • 40µl of the chloramphenicol stock for 20 ml
     • 20µl of the cyclohexamide stock for 20 ml
3. Incubate for 15 mins at 30°C.
4. Transfer to a 50 ml conical
5. Resuspend cells in 1 ml of SD Met- media
6. Transfer to an eppi tube
7. Wash 2x in SD Met- (1 ml each)
8. Resuspend in 1 ml of SD Met (w/ cyclohexamide) containing 225 µCi of ³H -Met (150 µCi per ml of culture)
   • Use 30 µl of 5 mCi/ml stock for 1 ml resuspension
9. Transfer the contents inside Eppi into a 5ml Falcon Conical tube with loose top (to allow pressure from air to be released in the incubateion step), slide the entire conical tube into another test tube and incubate on the rollerdrum at 30°C for 90 mins.
10. Pre-conjugate your antibody onto the proper beads – (for Npl3, used 50µl of Prot A sepharose (washed 3x 1ml with lysis buffer) with 2µl of anti-Npl3 antibody; in a 400 µl volume of lysis buffer, rotating at 4°C).
11. Wash 2 times in 1X PBS (1 ml each).
    • You can freeze pellet in FastPrep tubes at –80°C until another time to do your IP.
12. Complete your lysis buffer with proper protease inhibitor cocktail (PLAAC/PMSF or Roche’s ‘Complete’). Chill your lysis buffer on ice.
13. Lyse cell pellet in 100 µl of lysis buffer (PBSMT + 0.5M NaCl) for 30 sec at 6.5 setting in the bead beater – use one scoop of the acid-washed glass beads.
14. Add 400 µl of lysis buffer - incubate for 5 min on ice
15. Centrifuge for 10 mins. Remove the sup into a clean eppi, centrifuge for another 5 mins.
16. During the centrifugation, wash your antibody conjugated beads 2x with 1ml completed lysis buffer each time.
17. Add 400 µl of this clear lysate to the prot-A sepharose beads that have been pre-conjugated with anit-Npl3 antibody (or other antibody/beads combo).
18. Incubate for 2 hrs at 4°C.
    • Prepare 2 SDS-PAGE gels during this incubation.
19. Wash 3x with 1 ml of lysis buffer, remove as much buffer as you can.
20. Add the SDS-Loading buffer to the beads (40µl), boil for 5 mins. load all IP’ed material, add more loading buffer to the beads again, boil and load the 2nd gel for Western
21. After the gel is finished, the 1st loading gel will be used for coomassie staining (30 mins or longer, RT, shaking)
22. Destain – length of time depending on the commassie.
23. Remove the destain, transfer the gel to a clear fisher pipette tip box and add just enough “ENHANCE” for 1 hr with gentle shaking
24. Discard solution into appropriate waste jar in the hood, add dd-water for 30 mins, with gentle shaking.
25. Dry down the gel (on a Whatman paper) on a gel dryer for 1 hr.
26. Expose the gel to Kodak Biomax MS film at –80°C with intensifying screen; develop the film after 2-3 days and put another one on for a much longer exposure (weeks to months).