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Application of in‐gel protease assay in a biological sample: Characterization and identification of urokinase‐type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC‐3
Author(s) -
Zhao Zhenjun,
Raftery Mark J.,
Niu Xiao Mei,
Daja Merilla M.,
Russell Pamela J.
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305778
Subject(s) - proteases , protease , plasminogen activator , trypsin , blot , urokinase , gel electrophoresis , chemistry , microbiology and biotechnology , proteomics , biochemistry , prostate cancer , polyacrylamide gel electrophoresis , biology , cancer , enzyme , genetics , gene , endocrinology
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.