Application of in-gel protease assay in biological sample

Characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3

Zhenjun Zhao, Mark J. Raftery, Xiao Mei Niu, Merilla M. Daja, Pamela J. Russell

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.

Original languageEnglish
Pages (from-to)1142-1148
Number of pages7
JournalElectrophoresis
Volume25
Issue number7-8
Publication statusPublished - Apr 2004
Externally publishedYes

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