title: Active protease mapping in 2DE gels.
abstract: Proteases act as the molecular mediators of many vital biological processes. To understand the function of each protease, it needs to be separated from other proteins and characterized in its natural, biologically active form. In the method described in this chapter, proteases in a biological sample are separated under nonreducing conditions in 2DE gels. A specific small protease substrate, tagged with a fluorescent dye, is copolymerized into the SDS gel in the second dimension. After electrophoresis, the proteins are renatured by washing the gel with Triton X-100 solution or Milli Q water to remove SDS. The gel is then incubated in a protease assay buffer. The hydrolysis of the tagged specific substrate by the renatured protease releases the free fluorescent dye, which fluoresces in situ. The fluorescent spots indicate the location of the specific proteases in the gel and the specificity of the proteases.
authors.affiliation: Institute for Eye Research and Vision Cooperative Research Centre, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Zhenjun
authors.initials: Z
authors.lastname: Zhao
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Russell
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010447:Peptide Hydrolases