Novex® Zymogram Gels

Zymography is used for detecting and characterizing metalloproteinases, collagenases, and other proteases that can utilize casein or gelatin as a substrate. Novex® Zymogram Gels are excellent tools for detecting and characterizing proteases that utilize casein or gelatin as a substrate. Casein and gelatin are the most commonly used substrates (Figure 1) for demonstrating the activity of proteases. Novex® Zymogram Gels have been used to analyze a variety of enzymes, including matrix metalloproteinases, lipases, and other proteases [1-4].

Zymogram Gel Products

How Novex® Zymogram Gels Work

Protease samples are denatured in SDS buffer under non-reducing conditions and without heating, and run on a Novex® Zymogram Gel using Tris-Glycine SDS Running Buffer. After electrophoresis, the enzyme is renatured by incubating the gel in Zymogram Renaturing Buffer containing a non-ionic detergent. The gels are then equilibrated in Zymogram Developing Buffer (to add divalent metal cations required for enzymatic activity), and then stained and destained. Regions of protease activity appear as clear bands against a dark blue background where the protease has digested the substrate (Figure 1). Three different types of Novex® Zymogram Gels are available from Invitrogen. See Table 1 for specifications and selection of the gel types.


Figure 1. Zymography of proteins after gel electrophoresis. Following separation on a Novex® Zymogram Gelatin Gel (10%), proteins are renatured using Novex® Zymography Renaturing Buffer to allow substrate cleavage. Coomassie Blue® staining of gel results in clear area where substrate was digested by protease. Lanes 2-7: Serial dilution of Type IV collagenase 1.5 x 10^-5 units (7.8 ng).		click to enlarge Figure 2. The migration patterns of protein markers on Novex® Tricine, IEF, and Zymogram Gels are shown. Optimal resolution is achieved when protein bands migrate within the shaded regions. The numbered bands on the Zymogram Gel patterns refer to the following proteases: Band 1: Collagenase Type I (140 kDa); Band 2: Thermolysin (37 kDa); Band 3: Chymotrypsin (30 kDa); Band 4: Trypsin (19 kDa).

Table 1. Novex® Zymogram Gel types

	Novex® Zymogram Gelatin Gel	Novex® Zymogram Casein Gel	Novex® Zymogram Blue Casein Gel
Gel Composition	10% Tris-Glycine gel	12% Tris-Glycine gel	4–16% Tris-Glycine gel
Substrate	0.1% gelatin	0.05% casein	0.1% casein + Blue stain
Sensitivity	10^–6 units of collagenase	7 x 10^–4 units of trypsin	1.5 x 10^–3 units of trypsin
Post-staining Required?	Yes	Yes	No
Mol Wt Separation Range	20–120 kDa	30–150 kDa	10–220 kDa
Catalog No.	10 well (EC6175BOX) 12 well (EC61752BOX) 15 well (EC61755BOX)	10 well (EC6405BOX) 12 well (EC64052BOX)	10 well (EC6415BOX)

Resources for Novex® Zymogram Gels
Instruments & Accessories Electrophoresis Chambers Power Supplies & Accessories Western Blot Transfer Devices Western Automated Processing Instrument		Buffers & Reagents Protein Standards & Ladders Buffers & Reagents Protein Gel Staining & Imaging Western Blot Analysis Products		Technical Resources Gel Electrophoresis Protocols SDS-PAGE Protocols Novex® Pre-Cast Gel Electrophoresis Guide