Gelatin Zymography of Conditioned Media from Tumor Cell Lines.

Matrix metalloproteinases (MMPs) are involved in various processes related to tumor cells invasiveness. Since many of these cells can produce and secrete MMPs, using conditioned media from tumor cell cultures can be an effective method for assessing MMP proteolytic activity in vitro through zymography. This method utilizes a polyacrylamide gel with gelatin substrate. The separation of MMPs by electrophoresis is performed under denaturing but nonreducing conditions, ensuring that their proteolytic activity is preserved. After electrophoresis, the gel is incubated with a nonionic detergent and transferred to a buffer containing Ca. Protease activity is observed as clear zones against a uniformly blue background of nondegraded substrate stained with Coomassie blue.