Application of Gelatin Zymography in Nanotoxicity Research
Gelatin zymography is a relatively simple, inexpensive, and powerful technique to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It has been used particularly to detect the two members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. MMP-2 and MMP-9 are also able to degrade a number of extracellular matrix molecules including type IV, V, and XI collagens, laminin, and aggrecan core protein, thus making them important in the nanotoxicity research. In this technique, proteins are separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and gelatinases, activated by SDS, digest gelatin embedded in the gel. After staining with Coomassie Brilliant Blue R-250, areas of degradation are visible as clear bands against a blue-stained background. Here, we describe the detailed procedure for gelatin zymography.
Key wordsMatrix metalloproteinases MMP-2 MMP-9 Nanoparticles Zymography Gelatin
This work was partly supported by the NIESH/NIH (ES023693 and ES028911), KSEF-148-502-16-381, Kentucky Lung Cancer Research Program, and an Intramural Research Incentive Grants (50992) from UofL to Dr. Qunwei Zhang.
- 2.Murphy G, Willenbrock F, Crabbe T, O’Shea M, Ward R, Atkinson S, O’Connell J, Docherty A (1994) Regulation of matrix metalloproteinase activity. Ann N Y Acad Sci 732:31–41. https://doi.org/10.1111/j.1749-6632.1994.tb24722.xCrossRefPubMedGoogle Scholar
- 6.Wan R, Mo Y, Zhang X, Chien S, Tollerud DJ, Zhang Q (2008) Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: the role of oxidative stress and protein tyrosine kinase activation. Toxicol Appl Pharmacol 233(2):276–285. https://doi.org/10.1016/j.taap.2008.08.022CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Wan R, Mo Y, Chien S, Li Y, Li Y, Tollerud DJ, Zhang Q (2011) The role of hypoxia inducible factor-1α in the increased MMP-2 and MMP-9 production by human monocytes exposed to nickel nanoparticles. Nanotoxicology 5(4):568–582. https://doi.org/10.3109/17435390.2010.537791CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Oum’hamed Z, Garnotel R, Josset Y, Trenteseaux C, Lauren-Maquin D (2004) Matrix metalloproteinases MMP-2, -9 and tissue inhibitors TIMP-1, -2 expression and secretion by primary human osteoblast cells in response to titanium, zirconia, and alumina ceramics. J Biomed Mater Res A 68((1):114–122. https://doi.org/10.1002/jbm.a.20001CrossRefGoogle Scholar