Imidazole → Diethylstilbestrol

Participants

Reaction Steps

Cells of E. coli 7Δ harboring pELAC-MBP-DES-HT was grown in LB medium containing 100 μg/mL ampicillin in a test tube at 37° C. up to OD610nm˜2. 2 mL of the resulting culture was inoculated into 100 mL of LB medium supplemented with IPTG (final concentration of 0.1 mmol/L) in a 500 ml Sakaguchi flask at 30° C., for 8 hours. Induced cells were harvested from 1.6 L of cultivation broth, re-suspended in 200-240 mL of HT-II buffer (50 mM Tris-HCL, pH 8.0, 0.3 M NaCl, 10 mM imidazole, 15% glycerol), and sonicated using sonicator (INSONATOR 201M, KUBOTA). The debris was removed by centrifugation at 4° C. and 14,000 rpm for 15 minutes followed by filtration through 0.45 mm filter (Millipore). A solution of crude proteins was loaded onto HisTALON Superflow cartridge, 5 ml (Clontech) using AKTA avant 25 (GE Healthcare) in accordance with the manufacturer's recommendations. Fractions containing MBP fused His6-tagged DES were combined, and desalted using PD-10 columns (GE Healthcare) equilibrated with SC-buffer (50 mM Tris-HCl, pH8.0, 0.3 M NaCl, 15% glycerol).

Provenance & Evidence