Preparation of protein extracts for western blot
- Grow cells to mid-log (OD 600 less or equal to 1.0).
- 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).
- Pellet 3-5 min. in Tomy, 3K (large rotor, do not need adaptors).
- Wash 1X with 2 ml 50 mM Tris, pH 7.5., blot away excess liquid and freeze pellet on dry ice.
- To frozen pellet add 0.2g 0.5 mm glass beads and 50 µl 2% SDS.
- Vortex 90-120 sec full speed.
- Place in boiling water bath for 3 min.
- Add 100 µl 2x Laemmli sample buffer( or use 75 ul of 3X Laemmli sample buffer).
- Place in boiling water bath for 1 min.
- 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.
- 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.
- Load about 10-20 µl per minigel lane, depending on initial OD. For a preparative gel, load 150 ul into the large (common) lane.
