Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels

To begin, dissolve samples in conventional zymography sample buffer. Add 400 milliliters of 1x Tris-Glycine SDS running buffer to the gel apparatus. Load up to 35 microliters of sample per well. Run the samples at 120 volts at 4 degrees Celsius for 1.5 hours or until the molecular weight standards indicate that the proteases of interest are within the peptide resolving gel layer. After this, remove the gels from the plastic cassette.

Wash the gels three times in renaturing buffer at room temperature under gentle agitation, with each wash lasting 10 minutes. Transfer gels to a developing buffer solution for 15 minutes. Then, replace the solution with fresh developing buffer and incubate at 37 degrees Celsius under gentle agitation for 24 hours. After this, use a fluorescent gel scanner/imager with the appropriate excitation and emission filters to image the gels.