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Electrophoretic detection of chitinase isoenzymes using the PhastSystem
Author(s) -
McBride Jeffrey D.,
Stubberfield Colin R.,
Hayes David J.
Publication year - 1993
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.1150140126
Subject(s) - chitinase , chitin , isoelectric focusing , staining , isozyme , biochemistry , hydrolysis , chromatography , electrophoresis , fluorescence , chemistry , polyacrylamide gel electrophoresis , microbiology and biotechnology , biology , enzyme , chitosan , physics , quantum mechanics , genetics
Zymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter‐staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4‐methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4‐methylumbelliferone). The p I s of chitinase isoenzymes were estimated by using coloured p I markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii .