Active protease mapping in 2DE gels

Zhenjun Zhao*, Pamela J. Russell

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)431-438
Number of pages8
JournalMethods in molecular biology (Clifton, N.J.)
Publication statusPublished - 2009
Externally publishedYes


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