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 ?

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Petri Kursula           "I am somehow less interested in the weight and
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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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