1. Prepare sample. It has to be non-reducing conditions and excluded EDTA.
(CHO-cells e.g. should be cultured ca. 18h under serum-free conditions before using the culture media).
2. Make poly-acrylamide gel (7.5% final aclylamide concentration in the gel for MMP-9). Tom's proposal is to add 30mg gelatin to the 12 ml (i.e. 2.5 mg/ml). The general recommendation is a final concentration of 0.8 mg/ml gelatin in the gel. Gelatin is purchased from Sigma. Cat.# G-2625. Wear gloves (unpolymerized acrylamide is neurotoxic).
3. Apply samples and run gel. Use middle lanes preferably, because the side lanes usually deviate.
a) 5 µl of prestained SDS-PAGE standard (Biorad, broad range, Cat.# 161-0318),
b) 10.5 µl KT 1080 + 10.5 µl H2O + 7 µl 4x non-reducing buffer,
c) 21 µl sample (serum free condition media) + 7 µl non-reducing buffer, load with only 25 µl of this mix.
Run the gel for about 45 min (until the blue band reaches the bottom) at 200 V constant. Watch for loss of running buffer, refill if neccessary.
KT 1080 is derived from human fibrosarcoma cells and serves as a positive control for gelatinase B (92 kDa) and gelatinase A (72 kDa).
4. Wash the gel in zymography washing buffer (1 x TBS + 0.05%
Triton X-100) with constant shaking, at room temperature for 15
min, 10 min and 10 min. Wear gloves (skin enzymes produce
artifacts).
Triton X-100 (Octyl Phenoxy Polyethoxyethanol,
Sigma T-6878), a nonionic detergent, is used to replace SDS.
After Triton itself is removed by 3 washes with 1 x TBS, the
protein can refold and function.
5. Incubate the gel in zymography incubation buffer at 37°C for 1h - O/N. For RCHO-cells 18-20 h of incubation proved to be optimal.
6. Stain for Coomassie blue for 30 min.
7. Destain for 2-3 times.