Electrophoretic detection of chitinase isoenzymes using the PhastSystem

✍ Scribed by Jeffrey D. McBride; Colin R. Stubberfield; David J. Hayes

• Book ID: 102180493
• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 358 KB
• Volume: 14
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150140126

✦ Synopsis

Electrophoretic detection of chitinase isoenzymes using the PhastSystem

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 pls of chitinase isoenzymes were estimated by using coloured plmarkers. 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 Pneurnoqstis carinii.