Koshland Web Site/Methods
Preparation of protein extracts for western blot
  1. Grow cells to mid-log (OD 600 less or equal to 1.0).
  2. Harvest about 5 OD's of cells in a 13X100 glass dispo tube (Use new tubes. Can be up to about 6 mls of culture).
  3. Pellet 3-5 min. in Tomy, 3K (large rotor, do not need adaptors).
  4. Wash 1X with 2 ml 50 mM Tris, pH 7.5., blot away excess liquid and freeze pellet on dry ice.
  5. To frozen pellet add 0.2g 0.5 mm glass beads and 50 µl 2% SDS.
  6. Vortex 90-120 sec full speed.
  7. Place in boiling water bath for 3 min.
  8. Add 100 µl 2x Laemmli sample buffer( or use 75 ul of 3X Laemmli sample buffer).
  9. Place in boiling water bath for 1 min.
  10. Spin briefly in Tomy (at room temp--15 sec, 3K), remove liquid with a ml pippetteman tip, and transfer to 1.5 ml microfuge tube. Spin in microfuge for 10 min.
  11. Remove supernatant and transfer to new tube. This can be used immediately or frozen at -20oC. If frozen, boil sample for 3 min. prior to loading on gel.
  12. Load about 10-20 µl per minigel lane, depending on initial OD. For a preparative gel, load 150 ul into the large (common) lane.
Comments: Freezing samples prior to lysis is thought to reduce protein degradation!!
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