Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Journal Article · Mon Oct 16 00:00:00 UTC 2017 · Journal of Visualized Experiments

DOI: https://doi.org/10.3791/56424 · OSTI ID:1419440

Pidatala, Venkataramana R. ^[1]; Mahboubi, Amir ^[1]; Mortimer, Jenny C. ^[1]

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1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 1419440
• Journal Information: Journal of Visualized Experiments, Journal Name: Journal of Visualized Experiments Journal Issue: 128 Vol. 2017; ISSN 1940-087X; ISSN JVEOA4
• Publisher: MyJoVE Corp.Copyright Statement
• Country of Publication: United States
• Language: English