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].

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. See Table 1 for specifications and selection of the gel types.

Novex® Zymogram Gel

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).

Migration patterns of protein markers on Novex® Tricine, IEF, and Zymogram Gels

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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
Cat. No. 10 well (EC6175BOX)
12 well (EC61752BOX)
15 well (EC61755BOX)
10 well (EC6405BOX)
12 well (EC64052BOX)
10 well (EC6415BOX)

More information

See Novex® Zymogram gels and buffers in action in the following video on JoVE:

Protocol

References

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.