so called Zymogram technique has been used by many workers to identify
proteases from amoeba (mainly Acanthamoeba where the extra
cellular protease is implicated in corneal degradation).We have modified this method for use in the
"Mini-Protean" gel system by BioRad. From
the methods of Heussen & Dowdle, 1980, Kleiner & Stetler-Stevenson
1994, Mitra et al (1994).
It is convenient to use Fish Skin Gelatin (Sigma G-7765).
It is important that b-ME and DTT be kept out of any sample buffer for which protease
activity is to be detected. Samples
must therefore be made from non-reducing sample buffer:-
0.4M Tris pH6.8
(Bromephenol Blue added to give colour)
2). Make up gel mix including the gelatin at a suitable
concentration (0.1%). The
following is for
the BioRad ôminiproteanö system and is enough for two
Fish skin gelatin
3). Run gel at 200v as usual, and then incubate the gel with 2.5%
Triton X-100 ; 50mM Tris-HCl pH 7.0
for 1 hour and then overnight at in 50mM Tris-HCl pH 7.0, 2mM
CaCl2 at room temperature.
Coomassie blue Staining
overnight incubation, stain with Coomassie as normal:-
Rock the gel in the stain for about one hour, then destain by briefly
washing the gel in tap water and then rocking in destain solution:-
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