Coomassie blue r 250

After 7 days of methanol-induced culture, the cell-free culture supernatant was collected by centrifugation at 8000 rpm for 15 min. Then, the supernatant was precipitated with ammonium sulfate at 80% saturation and 4 °C overnight. The suspension was centrifuged at 8000 rpm for 15 min and the precipitate was dissolved in 20 mM phosphate buffer solution (pH 7.4). Afterwards, His-tagged recombinant enzymes were purified using Ni²⁺ affinity chromatography (HisTrap™ FF crude; GE Healthcare, Buckinghamshire, UK), as previously described [49 (link)]. Protein concentrations were estimated using a Pierce™ BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE analysis was carried out in a 12% (w/v) polyacrylamide gel, and staining was conducted with Coomassie blue R-250 (Sigma-Aldrich, St. Louis, MO, USA) and a Pierce™ Glycoprotein Staining Kit (Thermo Scientific), respectively. PNGase F, which is the most effective enzyme for specifically removing N-linked glycans (but not O-linked glycans) from glycoproteins [51 (link)], was obtained from New England Biolabs (Ipswich, MA, USA).

A total of 20 g of protein was present in an aliquot of the skimmed milk, which was then combined (1:1) with a sample buffer at pH 6.8 made up of 62.5 mM Tris-HCl, 25% glycerol, 4% SDS, and 0.01% bromophenol blue (Bio-Rad). After that, it was run through a 7.5% native SDS-PAGE (Minigel, Bio-Rad, California, USA) with 0.1% bovine gelatin (Sigma-Aldrich, Saint Louis, MO, USA) in the resolving gel. The gels were then incubated at 37°C for an overnight period in a 50 mM Tris-base buffer (Bio-Rad, California, USA) with pH 7.4 and 200 mM NaCl, 0.02% Brij-35, and 5 mM CaCl2 for gelatinolysis after 30 min at room temperature (25°C) in a renaturing solution of 2.5% (v/v) Triton X-100. The gels were stained for 30 min with 0.5% Coomassie Blue R-250 (Sigma-Aldrich) in 40% methanol, 10% glacial acetic acid, and 50% distilled water the following day. Next, they were destained for 30 min in 30% methanol, 7.5% glacial acetic acid, and 62.5% distilled water. Later, they were preserved in distilled water. Matrix metalloproteinase-2 and -9 were visible bands against the blue backdrop. After scanning with a CanoScan LiDE 120 Scanner (Canon Inc., Tokyo, Japan), it was integrated using ImageJ software version 1.80 (National Institute of Mental Health, Bethesda, Maryland, USA) [22 (link)]. The area of the band was used to represent the degree of gelatinase.

Cancer cell lysates (10 µg of each protein extract) were analyzed by 10% SDS-PAGE with Coomassie Blue R-250 staining (Sigma-Aldrich, St. Louis, MO, USA).
For immunodetection, 10 µg of each protein extract were run in parallel using 10% SDS-PAGE. Then, proteins were transferred to nitrocellulose membranes (Hybond-C extra) using semi-dry transfer (Bio-Rad, Hercules, CA, USA) [16 (link),17 (link)]. After blocking, membranes were incubated at optimized dilutions with alternatively antiPR monoclonal antibody (R&D Systems, Minneapolis, MN, USA) or anti-tubulin monoclonal antibody (Sigma) as loading control followed by incubation with SAv-HRP (R&D Systems, Minneapolis, MN, USA) at 1:1000 dilution or HRP-anti-mouse IgG (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) at 1:5000 dilution, respectively. Specific reactive proteins were visualized with SuperSignal West Pico Maximum Sensitivity Substrate (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).