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