Protein expression and purification
Growing expression Cultures
Growth of standard expression cultures (100 ml)
Culture growth for preparative production (1 liter)
- Inoculate 20 ml culture medium containing the relevant antibiotics. Grow overnight at 37°C with vigorous shaking.
- Inoculate a 1 liter culture 1:50 with the noninduced overnight culture. Grow at 37°C with vigorous shaking until an OD600 of 0.6 is reached.
Tip: Take a 1 ml sample immediately before induction. This sample is the noninduced control. Pellet cells and resuspend in 50 µl 5x SDS-PAGE sample buffer (see table 5x SDS-PAGE sample buffer). Store at –20°C until SDS-PAGE analysis.
- Induce expression (e.g., by adding IPTG to a final concentration of 1 mM).
- Incubate the culture for an additional 4–5 h.
Tip: Collect a second 1 ml sample. This is the induced control. Pellet cells in a microcentrifuge tube and resuspend in 100 µl 5x SDS-PAGE sample buffer. Store at –20°C until SDS-PAGE analysis.
- Harvest the cells by centrifugation at 4000 x g for 20 min. Freeze the cells in dry ice–ethanol or liquid nitrogen, or store cell pellet overnight at –20°C.