Bio safe coomasie stain

The recombinant plasmid (pET-malH) was transformed into E. coli Rosetta™ cells, and grown at 37 °C in 4 L of Luria Bertani broth (LB) containing 34 μg/mL chloramphenicol and 30 μg/mL kanamycin. When cultures reached late log phase (OD₆₀₀ of 0.6–0.8), they were induced with 1 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 h. Cells were harvested by centrifugation (4000 rpm × 20 min, at 4 °C), resuspended in sodium phosphate buffer (NaH₂PO₄, pH 8.0; 3 M NaCl, 10 mM imidazole), and lysed by sonication on ice (100 W, 1 s of sonication vs. 2 s pause, 500 cycles). The cell lysate was then centrifuged (13,000 rpm, 15 min, 4 °C). The resulting supernatant was loaded into a chromatography column packed with Ni-NTA agarose (Qiagen, Venlo, Germany), and washed with sodium phosphate at increasing imidazole concentrations of up to 80 mM. The elution was performed using sodium phosphate containing 250 mM of imidazole. Eluted fractions were tested for alpha-glucosidase activity as described in Section 2.4. The alpha-glucosidase containing fractions were resolved by Polyacrylamide Gel Electrophoresis (SDS-PAGE) using 10% polyacrylamide gels, stained with Bio-Safe™ Coomasie Stain (BioRad Inc., Hercules, CA, USA). Protein concentration was determined using the Pierce BCA Protein Assay (ThermoScientific Inc., Bridgewater, NJ, USA), using bovine serum albumin (BSA) as a standard.