- Zymography is a sensitive, semi-quantitative electrophoretic technique used to detect and analyze the enzymatic activity of proteases, especially matrix metalloproteinases (MMPs) like MMP-2 and MMP-9 (gelatinases).
- It is commonly employed in studies of epithelial-to-mesenchymal transition (EMT) to assess the functional activity—not just expression—of these enzymes, which play a key role in ECM degradation and cell invasion.
Zymography Procedure:
- Sample Preparation: Proteins are extracted from cell lysates or conditioned media (typically serum-free to avoid background activity) and mixed with non-reducing loading buffer.
- Gel Electrophoresis: Samples are run on SDS-PAGE gels copolymerized with a specific substrate, such as:
- Gelatin → for gelatinases (MMP-2, MMP-9)
- Casein → for caseinolytic proteases
- Fibrin → for fibrinolytic enzymes
- Renaturation: After electrophoresis, the gel is incubated in renaturation buffer (usually containing Triton X-100) to remove SDS and allow refolding of active enzymes.
- Development: The gel is then incubated in development buffer at 37°C, providing optimal conditions for protease activity.
- Staining: Gels are stained with Coomassie Brilliant Blue, and zones of enzymatic activity appear as clear bands against a dark blue background, indicating substrate degradation.
