Cell lysis (ProteaseMAX)

Lysis buffer

  • ProteaseMAX (from Promega: http://www.promega.com/resources/pro…ncer-protocol/) 1%
  • Ammonium Bicarbonate (50 mM) 90%
  • ACN 10%
  • Total Volume 1 mL
  • Note: We have found that since we do this fairly quickly, we do not need to add extra protease inhibitors. If you would like, you may of course also add protease inhibitors to the lysis buffer; I would recommend the Roche mini-Complete inhibitors and Roche PhosSTOP if you are interested in phosphoproteomics.

Procedure
(for 10 cm2 flask)

  1. Remove old media
  2. Rinse with PBS
  3. Add trypsin (0.5 mL); incubate at 37 °C for ~3–5 min
  4. Add 3 mL of PBS to the flask then take up all solution containing cells to tube
  5. Centrifuge at 950 rpm (~1000 rcf?) for 5 minutes (3 times); remove supernatant
  6. Add cold lysis buffer to the pellet (on ice); 3:1 lysis buffer to cell pellet; resuspend
  7. Remove to Eppendorf tube and spin
  8. Heat at 40 °C for 20 minutes
  9. 3× probe sonication: 60 sec, pause 90 sec, amplitude 30%. (Alternatively, sonicate in water bath for ~30 min.)
  10. Vortex, then centrifuge at ~13,000 rcf for 20 minutes
  11. Remove the supernatant to new tube

This method was contributed by David