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Recombinant Protein Purification for GST or His

Buffer A Buffer B Buffer C
50mM Tris pH=8.0 50mM Tris pH=8.7 50mM Tris pH=8.0
250mM NaCl 200mM NaCl 250mM NaCl
5mM EDTA 1mM EDTA
Add Fresh Add Fresh Add Fresh
10mM DTT 10mM DTT 5mM β2Me
1mM PMSF 1mM PMSF
  1. Weigh cell pellet then on ice resuspend in 2mL/g Buffer A- for GST or C- for Talon with lysozyme (1:1000 dil) and DNaseI (1:1000 dil) with 1mM PMSF + DTT - for GST or 5mM β2Me - for Talon.
  2. Sonicate 1X on ice, 10x pulse at 50% duty cycle, 5-6 output control.
  3. Add NaCl to 1M and sonicate 3x.
  4. Spin 25,000xg 30 min 4°C, save 10ul of lysate and 1ul pellet+9ul buffer for gel.
  5. Add 1:20 dil of 10% PEI (0.5% final) drop wise and mix 20 min at 4°C.
  6. Spin 25,000xg 30 min 4°C.
  7. Optional Precipitation: Add sat NH3SO4 to 65% drop wise while gently mixing, then spin 25,000xg 30 min 4°C. Weigh pellet and resuspend in 2mL/g Buffer A- for GST or C- for Talon without PMSF (for GST tag removal if desired).
  8. Add 1-2mL of slurry beads (Glut-Ag for GST or Talon for His) and rock for 2 hrs at 4°C. Glut-Ag 1mL beads (2ml slurry)=6ug protein, Talon1mL beads (2ml slurry)=3mg.
  9. Spin 1500 rpm 4°C 4 mins, save sup and wash beads, saving each wash:
    1. GST Glut-Ag - 3x Buffer A, 2x Buffer B, 2x Buffer B + 1M NaCl, add beads to column and wash through with Buffer B.
    2. Talon His - 4x Buffer C, 2X Buffer C + 1M NaCl, add beads to column and wash through with Buffer C. Can add 10mM imidazole to wash to get rid of non-specific binding.
  10. Elute with 500ul - 1mL fractions:
    1. GST Glut-Ag - Buffer B + 10mM glutathione, make 100mM glutathione fresh from power.
    2. Talon His - Buffer C + 150mM imidazole, make fresh from power.
  11. Check protein fractions with mini Bradford assay (2μl dye to 8μl elution), pool positive protein fractions and dialyze O/N at 4°C.

Download Protocol: Protein Purification
(note requires Microsoft Word to open)