Purificaton of TraI26

 

 

  1. Resuspend a 500mL cell pellet from the Ð80 C freezer with 50 mLs of 100mM 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 5mL Q column.  Load column with 100mM NaCl, 50mM 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 location of protein.  Protein should be in the flow through.  If the majority of the protein is not in the flow through, consider adjusting salt concentration to prevent it from sticking to the column.
  8. Apply flow through to a 5mL Blue column. Load column with 25mM NaCl, 50mM 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.
  9. Run selected fractions on a 10% SDS-PAGE to determine protein location and purity.
  10. IF GREATER PURITY  IS NEEDED: Raise NaCl concentration of selected fractions to ~3M.  Apply fractions to a 1mL Phe column. Load column with 3M NaCl, 50mM Tris (pH 7.5), 1 mM EDTA.  Collect flow through. Push protein off column with a gradient to 25mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA while collecting fractions.  This will increase the purity of the protein but result in a approx 50% loss of product. 
  11. Run selected fractions on a 10% SDS-PAGE to determine protein location and purity.
  12. If purity is sufficient, dialyze selected fractions into desired buffer, three changes, 8 hours each.