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.
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|
|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)|
See Novex® Zymogram gels and buffers in action in the following video on JoVE:
- Hu X, Beeton C (2010). Detection of Functional Matrix Metalloproteinases by Zymography. JoVE. 2010 45.
- Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science. 2010 Jan 22;327(5964):466-9.
- Ovchinnikova O, Robertson AK, Wågsäter D, Folco EJ, Hyry M, Myllyharju J, Eriksson P, Libby P, Hansson GK. T-cell activation leads to reduced collagen maturation in atherosclerotic plaques of Apoe(-/-) mice. Am J Pathol. 2009 Feb;174(2):693-700.
- Wegiel B, Bjartell A, Tuomela J, Dizeyi N, Tinzl M, Helczynski L, Nilsson E, Otterbein LE, Härkönen P, Persson JL. Multiple cellular mechanisms related to cyclin A1 in prostate cancer invasion and metastasis.J Natl Cancer Inst. 2008 Jul 16;100(14):1022-36.