Team:Washington/Notebook/50mL purification
From 2009.igem.org
Supernatant Protein Purification, 50mL
- Inoculate 50mL culture of TB with ~750uL overnight culture
- Grow a 37c until OD600: 0.4
- Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
- Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
- Transfer culture to 50mL Falcon Tube
- Centrifuge at 8000rpm for 20 min to pellet cells
- Pour supernatant into 60mL syringe with 0.45uM filter attached
- Apply filtered supernatant to Ni-column (see Ni-column Set up for directions)
- Collect flow through and re-apply to column
- Wash column with 12mL PBS
- Elute column into 15mL condenser tube with 12mL PBS with 100mM imidazole
- Spin elutant in condenser tube until desired volume (~500uL) this is you purified protein.