Team:Washington/Notebook/2mL purification


Uw title logo.png

Supernatant Protein Purification, 2mL

  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. Prep NiNTA columns
    1. Place micro-centrifuge columns in collection tubes
    2. In micro-centrifuge columns add 200uL NiNTA beads
    3. Add 500uL PBS, aspirate to thoroughly rinse columns
    4. Spin columns with collection tubes for 30sec at 500rpm
  6. Transfer 2mL of growing culture to eppendorf tube
    1. Spin tube for 20min at 8000 rpm
    2. Remove supernatant, carefully as to not disrupt the pellet and set aside
  7. Bind Protein to column
    1. Add 500uL supernatant to the column
    2. Spin for 30sec at 500rpm
    3. Discard flow through
    4. Repeat 1-3 until all supernatant has run though the column
  8. Wash column
    1. Apply 500uL PBS to column, aspirate to suspend beads
    2. Spin column for 30sec at 500rpm, discard flow through
    3. Repeat 1-2
  9. Elute protein off of column
    1. Make PBS with 1mg/mL BSA and 100uM imidazole
    2. Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
    3. Let sit for 2 min
  10. Place Column in clean collection tube
  11. Spin column for 5min at 500rpm