Preparation of protein extracts for western blot

1.Grow cells to mid-log (OD₆₀₀ 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 75μl 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 -20℃.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 150μl into the large (common)lane.