Large Scale Protein Expression & Purification

Reagents and Materials

• Autoclaved 1 L LB in 2.8 L flask
• Plates of protein producing bacteria being grown
• Shaker
• 1 mM IPTG
• Appropriate antibiotics
• Homogenizer
• Shaker
• AKTA
• Proteinase Inhibitor Cocktail
• Shaker
• 10mM HEPES buffer

Protocol

1. Culture protein-producing bacteria in the LB with appropriate antibiotics
2. Grow for 12 hours at 37°C and check OD
3. Induce with IPTG to a final concentration of 0.1mM when OD is 0.6
4. Let it grow for 4 hours at 30°C
5. Spin down the cells for 1 hour at 20 000 rpm
6. Homogenize the cells with a homogenizer. Add proteinase inhibitor cocktail
7. Take crude lysate and run it through the AKTA, an automatic His tag affinity chromatography machine.
8. Run the purified elutions on SDS-PAGE gel
9. Concentrate the proteins and store the proteins in 10 mM HEPES buffer, pH 8.0