User Manual

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12.4 Zymogram Gel Staining
Prior to staining zymogram gels, sample proteases must first be 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
Place 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.
Optimum conditions should be determined empirically. Stain gels with staining solution for at least 1 hr
at room temperature. Destain until clear bands appear against the blue background (~30–60 min).
12.5 TBE Gel Staining
Use Table 12.3 as a guide to selecting an appropriate staining method.
Table 12.3. TBE gel detection methods.
Method
Sensitivity
(Lower Limit) Advantages Disadvantages
Ethidium bromide 50 ng Classic fluorescent DNA stain Carcinogenic
Silver stain 1–2 ng More sensitive than ethidium bromide Requires multiple steps
SYBR
®
Green 0.02–2 ng High sensitivity Multiple steps, –20°C storage
SYBR
®
Safe 0.5 ng Non-hazardous Multiple steps
12.6 TBE-Urea Gel Staining
Use Table 12.4 as a guide to selecting an appropriate staining method.
Table 12.4. TBE-urea gel detection methods.
Method
Sensitivity
(Lower Limit) Advantages Disadvantages
Ethidium bromide 10 ng Classic fluorescent DNA stain Carcinogenic
Radiant
™
Red 10 ng Fast, single-step protocol RNA and ssDNA only
Silver stain 1–2 ng More sensitive than ethidium bromide Requires multiple steps
Criterion Precast Gels