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From 2010.igem.org

Protein purification

Materials:

- bacterial culture on plates

- LB medium

- 25 mM TEA (triethanolamine buffer), pH 8.0

- 1% lysozyme in TEA

- 4 M Urea

- 10% streptomycin in TEA

- 25 mM Tris buffer, pH 8.0

- glass beads

- centrifuge

- spectrophotometer

Protocol:

1. Cultivate bacterical cells on plates, 37 °C o/n (140 rpm)

2. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 250 mL LB medium containing the appropriate selective antibiotic. Incubate at 37°C with vigorous shaking

3. Induce production protein with 1 mM IPTG when OD600 is reached (check with 1.5 mL sample)

4. Harvest after 2 hours the 250 mL bacterial cells by centrifugation at 10.000 rpm for 10 min (Sorvall buckets)

5. Collect the free supernatant and store it

6. Wash pellet with TEA buffer, pH 8.0

7. Freeze pellet in -80 °C for 15 minutes

8. Resuspend pellet in 30 mL TEA with 1% lysozyme (final concentration 0.02%)

9. Incubate for 10 min at RT

10. Disrupt cells with glass beads and vortex for 10 minutes

11. Centrifuge at 14,000 rpm for 30 min at 4°C (protein is in supernatant)

12. Aliquot 1.8 mL supernatant into fresh eppendorf tubes and add 200 μL 10% streptomycin in TEA (final concentration 1%).

13. Centrifuge supernatant 14,000 rpm for 30 min at 4°C (protein is in pellet)

14. Resuspend pellet in 4 M Urea

15. Centrifuge supernatant 14.000 rpm for 30 min at 4°C (protein is in supernatant)

16. Dialyse supernatant from step 14 overnight 20 mM Tris buffer

17. Dialyse supernatant from step 5 overnight 20 mM Tris buffer