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|
- Weigh cell pellet then on ice resuspend in 2mL/g Buffer with lysozyme (1:1000 dil) and DNaseI (1:1000 dil) with 1mM PMSF +.
- Sonicate 1X on ice, 10x pulse at 50% duty cycle, 5-6 output control.
- Add NaCl to 1M and sonicate 3x.
- Spin 25,000xg 30 min 4°C, save 10ul of lysate and 1ul pellet+9ul buffer for gel.
- Add 1:20 dil of 10% PEI (0.5% final) drop wise and mix 20 min at 4°C.
- Spin 25,000xg 30 min 4°C.
- 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 without PMSF (for GST tag removal if desired).
- Add 1-2mL of slurry beads and rock for 2 hrs at 4°C. Glut-Ag 1mL beads (2ml slurry)=6ug protein, Talon1mL beads (2ml slurry)=3mg.
- Spin 1500 rpm 4°C 4 mins, save sup and wash beads, saving each wash:
- - 3x Buffer A, 2x Buffer B, 2x Buffer B + 1M NaCl, add beads to column and wash through with Buffer B.
- - 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.
- Elute with 500ul - 1mL fractions:
- - Buffer B + 10mM glutathione, make 100mM glutathione fresh from power.
- - Buffer C + 150mM imidazole, make fresh from power.
- 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
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