Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity
Published: April 30, 2023
Abstract
Source: Hu, X. et al., Detection of Functional Matrix Metalloproteinases by Zymography. J. Vis. Exp. (2010)
This video demonstrates the zymography gel electrophoresis technique to assess the activity of matrix metalloproteinases (MMPs) present in cell culture supernatant. The active MMPs degrade the MMP-specific substrate embedded in a polyacrylamide gel, which appears as clear bands against a dark stained background following staining.
Protocol
1. Loading and Running the Gel All samples must be prepared adequately to maintain the function of the enzymes and used immediately after collection or stored frozen at -80°C. The samples must not contain reducing agents (such a β-mercaptoethanol) or be boiled before gel loading. Open the pouch containing a gel inside a cassette, rinse the cassette with deionized water. Remove the protective tape from the bottom of the cassette and the comb from the top of the …
Offenlegungen
The authors have nothing to disclose.
Materials
| Xcell SureLock Mini-Cell CE mark electrophoresis apparatus | Invitrogen | EI0001 | |
| Power supply (model 302) | VWR | 93000-744 | |
| Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells | Invitrogen | EC61752BOX | |
| Blue Juice gel loading buffer | Invitrogen | 10816015 | |
| Gel loading tips | VWR | 53509-015 | |
| Protein molecular weight standard | Invitrogen | LC5800 | |
| Novex Tris-Glycine-SDS running buffer | Invitrogen | LC2675 | |
| Novex zymogram renaturing buffer | Invitrogen | LC2670 | |
| Novex zymogram developing buffer | Invitrogen | LC2671 | |
| SimplyBlue SafeStain | Invitrogen | LC6060 |
Diesen Artikel zitieren
Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity. J. Vis. Exp. (Pending Publication), e21120, doi: (2023).