Bio-Rad Ready Gel Precast Gels for 2-D Electrophoresis - Manual (Page 32)

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10.4 Zymogram Gel Staining
Prior to staining zymogram gels, sample proteases must be first renatured and allowed to break down the
substrate contained in the gel. The following protocol provides basic guidelines for detection. Optimal results
should be determined empirically.
Renaturing Solution
2.5% Triton X-100
Development Solution
50 mM Tris
200 mM NaCl
5 mM CaCl
2
(anhydrous)
0.02% Brij-35
Adjust to pH 7.5
Staining Solution
40% methanol
10% acetic acid
0.5% Coomassie Blue R-250
Destaining Solution
40% methanol
10% acetic acid
Proteins must be renatured first by placing the gels in renaturing solution for 30 min at room temperature.
Incubate gels in development solution at 37ºC for a minimum of 4 hr. Highest sensitivity is typically achieved
with overnight incubation. Optimal results should be determined empirically. Stain gels with Coomassie
Brilliant Blue R-250 staining solution for at least 1 hr at room temperature. Destain until clear bands appear
against the blue background, approximately
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