For you Developing buffer, which I call activity buffer I use  NaCl, CaCl2 and Tris-HCL and adjust the pH to 7.4 followed by overnight incubation of zymogram in this buffer at 37 degree. Make sure that you remove the SDS after running the gel, by treating the gel with 2.5% Triton-100 in water ( 100ml is sufficient for a 8X9cm gel) for at least 1 hour. This will help you in getting clear as well as better activity.

For cell extract pellet down the cells and incubate in RIPA buffer without EDTA, and reducing agent, add SDS  and detergent for sure,  incubate on ice for 30 min and then vortex followed by spinning at very high speed for 20 min at 4 degree. Collect the supernatant add the non reducing gel running dye and load the sample after quantitation. This will be very clear and will run smoothly on gel  also will be easily taken up by gel loading tips.

I would advice you to go for a low percentage gel say 8% and 1mg/ml of gelatin ( final concentration), run the gel for extended time so that there is clear separation to distinguish between pro- and active MMPs.

Hope this will work for you

Good Luck

Thank you very much for your reply. your protocol seems quite similar to the one I am currently using, but the increased TritonX-100 incubation time and the sample preparation before adding the gel running dye seem to be good improvements.

When you say spin at very high speed, could you provide a RPM number?

Once again, thanks.

I would advice you to go 12000rpm for 10 min or 8-10K for 20 min.

Please let me know if you need further assistance.

Thank you very much