Protocols

Co-IPP Protocol

  1. Grow S.pombe harboring insert in MM OD595=0.3-0.9 (mid-log) at 30ºC with agitation 260 rpm
  2. Collect 10 mL of OD595 0,8
  3. Adjust volume at 10 mL if cells are above 0,8
  4. Treatement with various stress conditions for 2 hours :
    • Heat shock 39ºC
    • Tunicamycin 10 ug/ml (Ci 10 mg/ml) at 30ºC 5 ul/5ml
    • DTT 10 mM (Ci 1M) at 30ºC 50 ul/5ml

All at 4ºC :

  1. Add directly NaN3 (Ci 1.538 M) to final concentration 10 mM (32.5 ul/ml)
  2. Spin in cold 5000 rpm 5 min.
  3. Suspend the pellet in 150 ul of ice-cold IPP Buffer (see above) plus pmsf (at 1 mM from 200 mM), IAA (10 mM from 0.5 M) and protease inhibitor cocktail (from x100, see above)
  4. Add 250 µl of glass beads and treat in beadbeater 4 times during 30 seconds or 10 times 30 seconds with the vibrobroyeur on maximal speed and leave 30 seconds on ice (for this use special Screwcap tubes)
  5. Add 400 ul of IPP Buffer plus
  6. Spin at13000 rpm for 5 min
  7. Remove 400 ul of the supernatant in a new tube
  8. Put 400 ul of IPP Buffer plus on the pellet and resuspend it gently
  9. Spin at13000 rpm for 5 min
  10. Take the supernatant (400 ul)
  11. Centrifuge (800 ul) 13000 rpm for 18 min and take the supernatant
  12. Add 50 ul of 10 % Protein A-sepharose. Slightly agitation 30-60 min at 4ºC (Pre-clearing step)
  13. Centrifuge 21 min at 13000 rpm, take the supernatant and spin 25 minutes again.
  14. Calculate protein concentration by Bradford.
  15. Add in a total vol of 800 ul, 2 ul of @calnexin (d=1/400) LAR224 (Preimm 204 or 205)
  16. Incubate one (1) hour at 4ºC with slight agitation
  17. Add 50 ul of Protein-A Sepharose (at 10% in IPP plus buffer) (1 g of protein A in 10 ml) and let it 1 hour at 4ºC
  18. Centrifuge 2 min at 13000 rpm, and remove the supernatant by aspiration
  19. Wash four times in 1 ml of IPP buffer (without protease inhibitor containing IAA and PMSF as above), during the wash centrifuge 5 min at 13000 rpm and put on rocker for 3 min
  20. Remove the supernatant and add 75 ul for big gel or 30 ul for small gel of Sample buffer 5X (see Bio Rad protocol) + 5% beta-Etoh. The buffer should contain the bromophenol blue.
  21. Boil the sample for 5 min, centrifuge 5 min at 12000 rpm, load the supernatant on protein gel.

Modified from a Co-IPP protocole of AM, PBB, PLT (07/10/2002)