Gelatin Zymography to Quantify Levels of MMP-2 and MMP-9 in Complex Biological Samples.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases associated with many disease states and aid in the degradation of the extracellular matrix (ECM). Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are gelatinases with many physiological functions. These proteases actively participate in many pathological states by cleaving the ECM and degrading other non-ECM substrates, such as tight junction proteins, cytokines and chemokines, growth factors, and adhesion molecules. Gelatin substrate zymography uses gelatin copolymerized in SDS-polyacrylamide gels to semi-quantitatively measure the enzymatic activity of gelatinases upon gel incubation in a developing buffer and subsequent gel staining. The study of MMP-2 and MMP-9 activity using gelatin substrate zymography is a simple and effective way to quantify MMP-2 and MMP-9 in a variety of samples. Here, we describe a protocol for detecting MMP-2 and MMP-9 using gelatin substrate zymography. We provide representative results using various sample types, including cell culture media, cell protein lysate protein, human and mouse plasma, and mouse brain protein lysate.