User Manual

Protease Analysis by
Zymogram PAGE
8
8.1 Introduction
Criterion
™
zymogram gels are used to test for proteolytic activity. Gels are cast with gelatin or casein,
which acts as a substrate for proteases that are separated in the gel under nonreducing conditions.
Proteases are detected by first renaturing the enzymes and then allowing them to break down the
substrate. Zymogram gels are stained with Coomassie Brilliant Blue R-250 stain, which stains the
substrate while leaving clear areas around active proteases.
8.2 Criterion Zymogram Gels
8.2.1 Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6, 0.2% NaN
3
Shelf life ~8 weeks at 2–8°C; expiration date is printed on each cassette
8.2.2 Gel Selection Guide
Zymogram Gel Optimum Separation Range
10% with gelatin 30–150 kD
12.5% with casein 20–120 kD
8.3 Zymogram Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffer (1x) 25 mM Tris, 192 mM glycine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH
2
O
Sample buffer 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 25% glycerol, 0.01% Coomassie
(catalog #161-0764) Brilliant Blue G-250
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