GST Fusion Protein Purification.docVIP

GST Fusion Protein Purification Stephen Helms 7/26/04 Materials 1X PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) Glutathione Elute Buffer (50 mM Tris-HCl, 10 mM Reduced Glutathione, pH 8.0) 75% Slurry Glutathione Sepharose Beads Procedure (adapted from Amersham’s) Prepare glutathione-sepharose beads as follows: Take 1.3ml 75% slurry of beads per mL desired 50% slurry (beads bind ~8mg/pelleted mL) Spin at 500g for 3 min Wash with 10 mL cold 1X PBS per mL desired slurry by mixing Spin at 500g for 3 min, then decant supernatant Add 1 mL 1X PBS per mL desired mL to make a 50% slurry Lyse cells Suspend cell pellet in the following: 1X cold PBS (50 μL/1 mL culture, or 50 mL for 1 L) 1 mg/mL Lysozyme [Not mentioned as standard component in Amersham] 25 mM PMSF (In 50 mL of 1X PBS add 2 mL 100 mM PMSF) [This is not in the Amersham procedure] Protease Inhibitor Cocktail (250 μL/50 mL 1X PBS) [Not in Amersham procedure] Sonicate cells in short bursts until disrupted Add 20% Triton x-100 to make 1% (for 50 mL, add 2.5 mL 20% triton or 500 μL 100% triton) Mix gently for 30 min to solubilize protein Spin at 12,000g for 10 min at 4°C (9-10k rpm, no comp. in JA-20 rotor) Bind protein to beads Add 50% slurry of beads to sonicated cells Incubate for 30 min at room temperature with gentle agitation Spin at 500g for 5 min to pellet beads Optional: Transfer to column and then continue, draining column instead of pelleting beads and pouring off supernatant Wash unbound protein from beads Add 10 bed volumes 1X PBS Spin at 500g for 5 min to pellet beads, pour off supernatant Repeat 2x to wash a total of 3x if batch washing Elute protein from beads Add 1.0 mL glutathione elution buffer per mL bed volume (add 0.154g glutathione per 50 mL elution buffer) Mix gently to resuspend if batch washing Incubate at room temperature for 10 min Spin at 500g for 5 min to pellet beads, collect supernatant Repeat 2x to elute 3x Notes Glutathione interferes with Lowry and BCA assays,