Materials

• 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

Protocol

1. Pellet an exponentially growing culture (typically, 10 ml at ~10⁶ cells ml^-1) at 1000 g for 10 minutes
2. Replace the supernatant with an equal volume of PBS and repeat centrifugation (1)
3. Discard the supernatant and resuspend the cell pellet in 500 μl PBS, and centrifuge in a microfuge for 2 minutes at 4500 g
4. 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
5. 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.