Detection of Gelatinases by Substrate Zymography

Zymography is a method of electrophoretic separation of matrix metalloproteinases (MMPs) in a polyacrylamide gel-containing substrate (called zymogram gel) for the assay of various MMPs in different biological samples. In particular, gelatin-zymography allows to determine simultaneously both active and latent forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in biological fluids as well as in tissue extracts with high sensitivity (order of pg) and in a semiquantitative manner.In this procedure, the proteins are separated by electrophoresis under denaturing but nonreducing conditions (to maintain enzymatic activity) in a polyacrylamide gel (SDS-PAGE) co-polymerized with a gelatin (denatured collagen) substrate. In the presence of SDS the enzymes are denatured exposing their active site, which permits both the latent and active forms of the gelatinases to exhibit gelatinolytic activity after their removal from the gel by a nonionic detergent (e.g., Triton X-100). After incubation in a calcium-containing buffer, the partially renatured enzymes can degrade the gelatin leaving a cleared zone that can be detected after staining of the gel. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a blue background of stained, undigested gelatin, that can be quantified by using an image analysis software.