Zymography is an electrophoretic technique used to detect hydrolytic enzymes and their activity. This method involves the use of polyacrylamide gel embedded with fluorogenic substrates to visualize protease activity through fluorescence.
Zymography is an electrophoretic technique that involves a protein substrate copolymerized with polyacrylamide gel to detect hydrolytic enzymes and their activity. To begin, dissolve samples in a zymography buffer containing glycerol, sodium dodecyl sulfate or SDS, and bromophenol blue.
Glycerol makes the sample buffer denser than the surrounding running buffer of the gel, enabling easy loading into the gel pockets. SDS - an anionic detergent - denatures enzymes in the sample and coats them with a negative charge. Bromophenol blue - a tracking dye - helps monitor the sample progress in the gel.
Load the samples in polyacrylamide gel embedded with fluorogenic substrates to detect protease activity. Now, connect the electrodes and begin electrophoresis. The generated electric field causes all the SDS-bound enzymes to migrate through the gel towards the positively charged electrode.
Following completion of electrophoresis, remove the gel and wash it in a renaturing buffer containing non-ionic detergent that displaces SDS to allow enzyme renaturation. Next, incubate the gel in a developing buffer containing divalent cations that activate the enzymes in the gel.
The active proteases cleave the fluorogenic substrates, causing an increase in the fluorescence. Image the gel to observe fluorescent protein bands whose intensity is proportional to protease activity.
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.
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