Protein expression and purification

Growing expression Cultures

  1. Inoculate 10 ml culture medium containing relevant antibiotics in a 50 ml flask. Grow the cultures overnight at 37°C.
  2. Inoculate 100 ml prewarmed medium (with antibiotics) with 5 ml of the overnight culture and grow at 37°C with vigorous shaking until an OD600 of 0.6 is reached (30–60 min).

    Tip: Take a 1 ml sample immediately before induction. This sample is the noninduced control. Pellet cells and resuspend them in 50 µl 5x SDS-PAGE sample buffer (see table 5x SDS-PAGE sample buffer). Store at –20°C until SDS-PAGE analysis

  3. Induce expression (e.g., by adding IPTG to a final concentration of 1 mM).
  4. Incubate the cultures for an additional 4–5 h.

    Tip: Collect a second 1 ml sample. This sample is the induced control. Pellet cells and resuspend them in 100 µl 5x SDS-PAGE sample buffer. Freeze and store the sample at –20°C until SDS-PAGE analysis.

    Tip: If expressing a protein for the first time, take a 1 ml sample every hour and treat as above to produce a time-course of expression

  5. Harvest the cells by centrifugation at 4000 x g for 20 min.
  6. Freeze the cells in dry ice–ethanol or liquid nitrogen, or store cell pellet overnight at –20°C
5x SDS-PAGE sample buffer
Composition of working solution Component  Amount per liter
0.225 M Tris·Cl (pH 6.8) 1 M Tris·Cl, pH 6.8 2.25 ml
50% glycerol Glycerol 5 ml
5% SDS SDS 0.5 g
0.05% bromophenol blue Bromophenol blue 5 mg
0.25 M dithiothreitol (DTT)* 1 M DTT 2.5 ml

  1. Inoculate 20 ml culture medium containing the relevant antibiotics. Grow overnight at 37°C with vigorous shaking.
  2. 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.

  3. Induce expression (e.g., by adding IPTG to a final concentration of 1 mM).
  4. 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.

  5. 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.
X
Cookies help us improve your website experience.
By using our website, you agree to our use of cookies.
Confirm