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The first step in protein purification is to express the protein in a cellular host. In our case we will be using E. coli. The pET28-His6-GFP construct we made contains an IPTG-inducible His6-GFP. Once the cells start growing, we will “induce” them to make the protein by adding IPTG. Then we will allow them to continue growing and producing the protein for several hours before harvesting the cells via centrifugation.
- 1mL overnight culture of cells containing BL21 cells transformed with pET28-His6-GFP
- 1000x kanamycin stock
- 1M IPTG stock
- 50mL of liquid LB media
- Add 1000X kanamycin to 50mL of LB so that the final concentration is 1X. Swirl to mix.
- Dilute the overnight culture 1:100 into 50mL of LB supplemented with 1X kanamycin. Shake culture at 37°C. Remove 1mL of sample periodically to check the OD600 (to measure the concentration of bacteria). See Appendix for Spectrophotometer usage instructions.
- When the OD600 reaches between 0.6-1.0 (approximately 2 hours), add IPTG to a final concentration of 500µM. This will induce the expression of the His6-GFP protein.
- Allow the culture to grow and express the GFP by shaking at 37°C for 16-24 hours. Note that in some cases, lowering the temperature after the induction may help proteins fold.
- After 16-24 hours, pour the culture into a 50mL conical tube.
- Pellet cells by spinning in a centrifuge at 4000 g for 10 minutes. Remember to balance the centrifuge!
- Remove supernatant by pouring into a waste container for liquid media (near the sink). We will bleach the media to ensure any bacteria left in the media are killed before disposing down the drain.
- Freeze the cell pellet until you’re ready to start the protein purification.