Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity

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

1. 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.
2. Open the pouch containing a gel inside a cassette, rinse the cassette with deionized water.
   1. Remove the protective tape from the bottom of the cassette and the comb from the top of the gel.
   2. Rinse the wells three times with running buffer (diluted to 1X in deionized water).
   3. Place the gel into the Mini-Cell, ensur....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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