GST Fusion Protein Purification
Stephen Helms
7/26/04
Materials
Procedure (adapted from Amersham’s)
1. Prepare glutathione-sepharose beads as follows:
a. Take 1.3ml 75% slurry of beads per mL desired 50% slurry (beads bind ~8mg/pelleted mL)
b. Spin at 500g for 3 min
c. Wash with 10 mL cold 1X PBS per mL desired slurry by mixing
d. Spin at 500g for 3 min, then decant supernatant
e. Add 1 mL 1X PBS per mL desired mL to make a 50% slurry
2. Lyse cells
a. Suspend cell pellet in the following:
i. 1X cold PBS (50 μL/1 mL culture, or 50 mL for 1 L)
ii. 1 mg/mL Lysozyme [Not mentioned as standard component in Amersham]
iii. 25 mM PMSF (In 50 mL of 1X PBS add 2 mL 100 mM PMSF) [This is not in the Amersham procedure]
iv. Protease Inhibitor Cocktail (250 μL/50 mL 1X PBS) [Not in Amersham procedure]
b. Sonicate cells in short bursts until disrupted
c. Add 20% Triton x-100 to make 1% (for 50 mL, add 2.5 mL 20% triton or 500 μL 100% triton)
d. Mix gently for 30 min to solubilize protein
e. Spin at 12,000g for 10 min at 4°C (9-10k rpm, no comp. in JA-20 rotor)
3. Bind protein to beads
a. Add 50% slurry of beads to sonicated cells
b. Incubate for 30 min at room temperature with gentle agitation
c. Spin at 500g for 5 min to pellet beads
4. Optional: Transfer to column and then continue, draining column instead of pelleting beads and pouring off supernatant
5. Wash unbound protein from beads
a. Add 10 bed volumes 1X PBS
b. Spin at 500g for 5 min to pellet beads, pour off supernatant
c. Repeat 2x to wash a total of 3x if batch washing
6. Elute protein from beads
a. Add 1.0 mL glutathione elution buffer per mL bed volume (add 0.154g glutathione per 50 mL elution buffer)
b. Mix gently to resuspend if batch washing
c. Incubate at room temperature for 10 min
d. Spin at 500g for 5 min to pellet beads, collect supernatant
e. Repeat 2x to elute 3x
Notes
1. Glutathione interferes with Lowry and BCA assays, but
Regeneration of Glutathione Sepharose 4B Beads
Stephen Helms
8/4/04
Materials
· 0.1 M Tris-HCl + 0.5 M NaCl, pH 8.5
· 0.1 M NaAcetate + 0.5 M NaCl pH 4.5
· 1X PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)
· 6 M Guanidine HCl
· 70% Ethanol
· 20% Ethanol
Procedure (from the Amersham procedure)
1.
a. Wash with 2 bed volumes 0.1 M Tris-HCl + 0.5 M NaCl, pH 8.5
b. Wash with 2 bed volumes 0.1 M Na-Acetate + 0.5 M NaCl, pH 4.5
c. Repeat 3-4 times
d. Re-equilibrate with 3-5 bed volumes 1X PBS
2. Remove precipitated and denatured proteins
a. Wash with 2 bed volumes 6 M guanidine HCl
b. Immediately wash with 5 bed volumes 1X PBS
3. Remove hydrophobic substances
a. Wash with 3-4 bed volumes 70% ethanol (alternatively, use 2 bed volumes 0.1% non-ionic detergent)
b. Immediately wash with 5 bed volumes 1X PBS
4. For long-term storage
a. Wash 2x with 10 bed volumes 1X PBS
b. Wash 2x with 10 bed volumes 20% ethanol
c. Store at 4°C
生物谷网站 http://www.bioon.com
