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Use of Gel Zymography to Examine Matrix Metalloproteinase (Gelatinase) Expression in Brain Tissue or in Primary Glial Cultures

2011; Springer Science+Business Media; Linguagem: Inglês

10.1007/978-1-61779-452-0_15

1940-6029

Harald Frankowski, Yu-Huan Gu, Ji Hoe Heo, Richard Milner, Gregory J. del Zoppo,

Blood Coagulation and Thrombosis Mechanisms

Glia synthesize, package, and secrete several species of matrix proteases, including the gelatinases (pro-)MMP-2 and (pro-)MMP-9. In appropriate settings (e.g., experimental ischemia), these MMPs can be assayed from cerebral tissues or from astrocytes and microglia in culture by enzymatic substrate-dependent assays and by gelatin-based zymography. We describe the methodologies for the sensitive quantitative development of the inactive and active forms of both MMP-2 and MMP-9 from tissues and cells, by means of lysis of the collagen substrate in collagen-impregnated gel electropheresis by the zymogen and active gelatinases. These methodologies are a refinement of those used commonly, with instructions to increase sensitivity. Serious and often overlooked issues regarding sources of sample contamination and elements confounding the MMP band development and their interpretation are discussed.