2.7. Gelatinase zymography

The gelatinase zymography is an electrophoretic technique used to measure the artificial proteolytic activity of enzymes separated in polyacrylamide gels under non-reducing conditions. The gelatinase zymography protocol was adapted from Rosenberg et al. (1995) and Murnane et al. (1997).

Fresh, non-perfused cortical tissue was homogenized in ddH₂O and exposed to 3 freeze-thaw cycles using a methanol dry ice bath and a 37 °C water bath to lyse cells. Samples were centrifuged at 12,000 rpm for 40 min to pellet insoluble material. A Bradford protein assay (BioRad, Hercules, CA) was used to quantify the amount of protein in the supernatant and 1000 μg of total protein of each sample was incubated with gelatin sepharose beads (GE HealthCare, Piscataway, NJ) to isolate gelatinases. Gelatinases were bound to the beads, washed and eluted from the beads using 20 mM phosphate buffer +10% DMSO. Isolated gelatinase samples were mixed with non-reducing 2× SDS loading sample buffer (Invitrogen, Carlsbad, CA), allowed to sit at room temperature for 5 min, and equal volumes were loaded into wells of Novex 10% Gelatin Zymogram gel (Invitrogen).

Following electrophoresis at 4 °C, the gel was removed, washed with 2.5% Triton 2 times, each for 30 min, and rinsed with ddH₂O 2 times, each for 15 min, all at 4 °C. The enzymes embedded in the gel were then activated by incubating the gel in a developing solution consisting of 50 mM Tris-HCl, pH 7.5; 5 mM CaCl₂; 0.15 M NaCl; 1 μM ZnCl₂; and 0.02% NaN₃ for ~72 h at 37 °C. The gel was stained with a 1% Amido Black solution in 50% MeOH+10% acetic acid for 10 min and de-stained in a 50% MeOH+10% acetic acid solution, until bands of clearing were observed. Areas of clearing indicated active gelatinases in the gel. A digital camera was used to capture images of gels and the areas of clearing at 95 and 92 kDa, that of pro-MMP-9 and active MMP-9, respectively, were quantified using Multi Gauge software (FujiFilm Corp. Life Science Division, Tokyo, Japan).