4.16. Gelatin Zymography for MMP-2 and MMP-9 Secretions Assay

After the cells were treated with or without HCT and PR at non-toxic concentrations (lower than IC20) in serum-free medium, the supernatant was collected. Protein concentrations were determined using the Bradford method. Then, 10 µg of protein samples were mixed with a substrate gel sample buffer (13.3% SDS, 40% glycerol, 42 mM Tris-HCl (pH 6.5), 0.013% bromophenol blue, reagent grade gelatin (1 mg/mL)) and loaded onto the gel without prior boiling. Gels were washed and incubated in Zymo buffer for 24 h. After that, the gels were stained with 0.5% Coomassie blue and then de-stained using de-staining buffer (methanol: acetic acid: water, 4:1:5). Areas of protease appeared as clear bands against a dark blue background where the metalloproteases had digested the gelatin [70].