One- and Two-Dimensional Zymography Methods for Assessing the Family of Proteases.

Zymography is a very powerful yet simple and cost-effective technique that is based on the electrophoretic separation and in-gel detection of enzyme activity using a repertoire of substrates. Zymography characterizes proteases and some other hydrolytic enzymes after running them on polyacrylamide gels under differing conditions, such as one- and two dimensional gels, immobiline pH gradient (IPG) strip, and enzymoblotting or enzyme overlay membrane (EOM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography helps determine the molecular weight of the proteases, enabling the detection of multiple forms of the enzyme or catalytic active monomers from complex mixtures. SDS-PAGE zymography is especially useful for the detection of proteases that naturally occur in complexes with protease inhibitors, since these complexes dissociate during the process of electrophoretic separation. Conversely, native zymography helps characterize the multimeric catalytic active forms that can be irreversibly denatured using SDS. Zymography after IPG strip and two-dimensional electrophoresis (2DE) can resolve complex biological samples on the basis of isoelectric point (pI), molecular weight, solubility and relative abundance, theoretically separating over 10,000 proteins. This chapter examines various zymography techniques that can facilitate the simple detection and characterization of proteases from both prokaryotic and eukaryotic sources because of their selective advantage over other protease characterization approaches.