Gelatin zymography: help!

[Luke Newman]

Hello all,

I'm trying to detect gelatinase expression in mammalian cells by
zymography, without any success so far. The set-up:

1) HT-1080 fibrosarcoma cells. Serum-free conditioned medium collected
   for 24 h, mixed with SDS gel loading buffer (no reducing agent nor
   heating).

2) 10% acrylamide gels containing 1 mg/ml gelatin (Sigma, type B from
   bovine skin).

3) Standard electrophoresis conditions, gel washed extensively in 2.5%
   Triton X-100 and then incubated in a neutral buffer containing some
   NaCl, calcium ions and Brij overnight at 37 degC.

4) Standard Coomassie R staining and destaining.

I get no bands at all, even though this cell line is often used as a
positive control and my method is essentially standard. I've tried
loading the maximum amount of conditioned medium that I can get in a
well and also various sources of Triton X-100. Could the type and/or
source of the gelatin be causing my complete lack of success ? Does
the panel have any other tips that might help ?

Many thanks in advance

Luke Newman

e.l.newman at dundee.ac.uk