Purification of TraI30, 302 amino acids

 

 

  1. Resuspend a 500mL cell pellet from the Ð80 C freezer with 50 mLs of 50mM NaCl, 50mM Tris (pH 7.5), 1 mM EDTA.
  2. Add 50uL of 100mM PMSF in 200 proof EtOH.
  3. Soniate in metal sonicator cup at max watts 4X by one minute with 2 minute breaks on ice.
  4. Add 50uL of 100mM PMSF in 200 proof EtOH.
  5. Spin out cell debris by centrifugation at 10000g for 10 minutes. Collect supernatant.
  6. Apply supernatant to a 5mL heparin column.  Load column with 50mM NaCl, 50 Mm Tris (pH 7.5), 1 mM EDTA.  Collect flow through.  Push protein off column with a gradient to 2M NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA while collecting fractions. 
  7. Run selected fractions on 10% SDS-PAGE to determine putiry and location of TraI30.
  8. Pool selected fractions from heparin column and dilute down to ~100 mM NaCl.  Load diluted fractions onto a Blue column with 100 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA.  Collect flow through.  Push protein from column with 2M NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA while collecting fractions.
  9. Run selected fractions on 10% SDS-PAGE to determine purity and location of TraI30. 
  10. If purity is sufficient, dialyze selected fractions into desired buffer, three changes, 8 hours each.