- 1x Sample buffer (62 mM Tris [pH 7], 10% glycerol, 2.3% SDS, 5% β-mercaptoethanol, bromophenol blue [the ‘end of a 200 ul tip’ is enough for 10 ml sample buffer]) store at -20°C
- Pellet an exponentially growing culture (typically, 10 ml at ~106 cells ml-1) at 1000 g for 10 minutes
- Replace the supernatant with an equal volume of PBS and repeat centrifugation (1)
- Discard the supernatant and resuspend the cell pellet in 500 μl PBS, and centrifuge in a microfuge for 2 minutes at 4500 g
- Remove as much of the supernatant as possible, and resuspend the cell pellet in 1x sample buffer to a final concentration of 105 cells per μl
- Vortex the cell lysate for ~2 seconds and place at 100°C for 2 min; centrifuge briefly (~2 seconds), and store at -20°C for up to 3 months
NB: if protein of interest is likely to be membrane-associated don’t boil, instead resuspend in 1x sample buffer containing protease inhibitors; alternatively, follow the hypotonic lysis protocol to assess the distribution of your protein between soluble and membrane fractions.