Team:Washington/Notebook/50mL purification


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Supernatant Protein Purification, 50mL

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