Zymographie

Zymography is an electrophoretic technique used to detect enzyme activity in a sample by incorporating a substrate directly into a polyacrylamide gel. The most common form, gelatin zymography, detects proteases that degrade gelatin, notably matrix metalloproteinases (MMPs). Other substrates such as casein or collagen enable visualization of different proteases.

In a typical protocol, proteins are separated by SDS-PAGE in a gel containing the chosen substrate. After electrophoresis, the gel is treated to remove SDS and renature the enzymes, usually by washing with a non-ionic detergent or renaturation buffer. The gel is then incubated in a development buffer that supports enzymatic activity, allowing active proteases to digest the embedded substrate. The gel is stained; areas where substrate has been degraded appear as clear bands against a stained background. The position of bands corresponds to the molecular weight of the active enzyme, and band intensity provides a semi-quantitative measure of activity.

Zymography is semi-quantitative and often semi-quantitative analysis is performed by densitometry, typically using standards or internal controls. Variants exist, including casein zymography and collagen zymography, which use different substrates to target specific enzyme classes. There are also non-denaturing approaches that minimize denaturation to preserve native activity.

Applications include studying tissue remodeling, cancer invasion, inflammatory diseases, and responses to protease inhibitors. Zymography can be applied to cell lysates, conditioned media, and tissue extracts. Limitations include dependence on substrate choice, potential activation of latent forms during renaturation, and limited quantitative precision compared with pure biochemical assays. The method originated in the 1980s and has since become a standard tool for profiling protease activity.