Detection of Amoebal Proteases

Detection of Amoebal Proteases
The 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). 1). 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:- *Working Concentration Stock 100mls* 20% Glycerol - 20g 5% SDS - 5g 0.4M Tris pH6.8 0.5M 80mls (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 “miniprotean” system and is enough for two gels:- Stock solutions 8% 10% 12% 14% Distilled Water 4.64mls 4.31mls 3.31mls 2.64mls 1.5M Tris pH8.8 2.5mls 2.5mls 2.5mls 2.5mls 10% SDS 100µl 100µl 100µl 100µl 45% Fish skin gelatin 22µl 22µl 22µl 22µl 30% Acrylamide mix 2.67mls 3.0mls 4.0mls 4.67mls 10% APS 50µl 50µl 50µl 50µl TEMED 20µl 20µl 20µl 20µl Total 10mls 10mls 10mls 10mls 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 After overnight incubation, stain with Coomassie as normal:- 0.1% Coomassie R-250 40% Methanol 10% Acetic acid 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:- 10% Acetic acid 5% Methanol
	Figure 1. Equal quantities of material from culture supernatants of various Acanthamoeba were added to the wells of a 10% gel containing 0.1% fish skin gelatin. The gel was incubated in triton buffer for 1 hour to remove SDS. Staining was by usual Coomassie blue method.
Another method that is useful for certain applications utilizes the fact that unprocessed film has a gelatinous backing whose digestion can be assayed (Cheung et al, 1991). This method is especially useful for detecting pH optima (Buckwold et al, 1999) and inhibitors.

Figure 2. This method is simply to take a strip of untreated film and to pipet on equal volumes of solution containing the protease and to incubate at a suitable temperature in a moist environment (so that the drop does not evaporate. After a suitable time (try an hour at first?) the film is gently washed under flowing water and the gelatin coat very gently but equally rubbed to reveal activity.

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