Gelatin zymography: help!

Petri Kursula pkursula at cc.oulu.fi
Fri Jan 22 04:57:52 EST 1999

I think you might need some Zn ions in the incubation medium. My protocol
has ZnCl2 at 1 micromolar. I don't know if it's essential though. But
gelatinases are metalloproteases, and I have the vague idea that they
require Zn for activity, at least they bind it. 

> 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 ?

Petri Kursula           "I am somehow less interested in the weight and
University of Oulu    convolutions of Einstein's brain than in the near
Petri.Kursula at oulu.fi  certainty that people of equal talent have lived
http://start.at/MAG          and died in cotton fields and sweatshops."
http://cc.oulu.fi/~pkursula                        -- Stephen Jay Gould 

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