Jhdavis::ClpXPrep

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  1. Express from BLR (pACYC-derived plasmid) at RT for 3 hours post-induction (induce at OD = 0.8 after cooling to RT for 25 mins)
  2. Lyse in 20 mM Hepes, 400 mM NaCl, 100 mM KCl, 20 mM Imidazole, 10% glycerol pH 7.6
  3. Lysozyme + french press with PMSF (generates foam in french press
  4. Spin down, add sup to 4 mL Ni-slurry
  5. 2 washes with lysis buffer+PMSF on bench, add to column
  6. 3x washes in column, with 1 wash used to resuspend the column
  7. 0.5 mL elutions with lysis buffer - PMSF + 250 mM Imidazole
  8. pool fractions by bradford
  9. S300 in 50 mM Tris, 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10%glycerol pH 7.5
  10. Check by gel
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