Coomassie brilliant blue r 250 cbb

Samples of the cell lysates taken before and after IPTG induction were suspended and boiled in 5× Laemmli sample buffer (Bio-Rad, USA), and examined by polyacrylamide gel electrophoresis (PAGE; Bio-Rad, USA) in the presence of SDS on 12.5% (w/v) gels (Laemmli, 1970 (link)). Following this, Coomassie brilliant blue R-250 (CBB; Bio-Rad, USA) was used to stain the gel before viewing.

Zymography was performed using Novex 10% Zymogram Gelatin Gel (Life Technologies/Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) or Precast Casein Zymogram Gel (Cosmo Bio Co., LTD., Tokyo, Japan). Samples were dissolved in NuPAGE LDS sample buffer (Invitrogen), and electrophoresis was performed at 30 mA for approximately 1 h with Novex Tris Glycine SDS running buffer (Life Technologies/Invitrogen/Thermo Fisher Scientific). The gel was shaken gently in 2.5% Triton X-100 solution for 1 h at room temperature with one buffer change, and incubated overnight with or without 10 mM EDTA in 50 mM Tris–HCl (pH 7.4) containing 10 mM CaCl₂. Proteinase activities were visualized as unstained bands after the gel was stained with Coomassie Brilliant Blue R-250 (CBB) (Bio-Rad Laboratories, Hercules, CA, USA). The apparent molecular weights of the protein bands were estimated by comparison with DynaMarker Protein MultiColor III (BioDynamics Laboratory Inc., Tokyo, Japan).

After IPTG induction, the bacteria cultures were harvested and the pellets were suspended in 2× Laemmli sample buffer (Bio-Rad, USA). The samples were boiled for 5 min before loaded into 12.5% (w/v) polyacrylamide gel electrophoresis system (PAGE, Bio-Rad, USA) in the presence of sodium dodecyl sulfate (SDS) according to the methods established by Laemmli (1970) (link). To identify the protein bands, the gels were stained with Coomassie brilliant blue R-250 (CBB; Bio-Rad, USA).