Preparation of Cell Lysate

1.Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS.Wash non-adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells.

2.Add ice-cold to cells (1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x106 cells/60 mm dish/75 cm2 flask).

3.Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water.Transfer the cell suspension into a centrifuge tube.Gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells.

4.Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes.Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.

5.Dilute the cell lysate at least 1 : 10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent.At this step,the sample can be divided into aliquots and stored at -20 for up to a month.

TIP: When working with large volumes of non-adherent cells,the cells may not be cooled quickly enough to maintain the activity of the protein being studied.In this case,pour the cell suspension into a mixture of an equal mass of 2 x PBS and ice,then collect the cells by centrifugation and perform the lysis as described above.