Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Pidatala, Venkataramana R.; Mahboubi, Amir; Mortimer, Jenny C.

doi:10.3791/56424

Title: Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

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Abstract

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.

Authors:

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

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

Publication Date:: 2017-10-16

Research Org.:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:: 1419440

Grant/Contract Number:: AC02-05CH11231

Resource Type:: Accepted Manuscript

Journal Name:: Journal of Visualized Experiments

Additional Journal Information:: Journal Volume: 2017; Journal Issue: 128; Journal ID: ISSN 1940-087X

Publisher:: MyJoVE Corp.

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; Biochemistry; Issue 128; Gel electrophoresis; polysaccharides; glycosyl hydrolases; mannanases; mannan; glucomannan; cell wall; hemicellulose; biofuels

Citation Formats


                    Pidatala, Venkataramana R., Mahboubi, Amir, and Mortimer, Jenny C. Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE.  United States: N. p., 2017. 
Web.  doi:10.3791/56424.

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                    Pidatala, Venkataramana R., Mahboubi, Amir, & Mortimer, Jenny C. Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE.  United States.  https://doi.org/10.3791/56424

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                    Pidatala, Venkataramana R., Mahboubi, Amir, and Mortimer, Jenny C. Mon .  
"Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE".  United States.  https://doi.org/10.3791/56424.  https://www.osti.gov/servlets/purl/1419440.

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@article{osti_1419440,

  title        = {Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE},

  author       = {Pidatala, Venkataramana R. and Mahboubi, Amir and Mortimer, Jenny C.},

  abstractNote = {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.},

  doi          = {10.3791/56424},

  journal      = {Journal of Visualized Experiments},

  number       = 128,

  volume       = 2017,

  place        = {United States},

  year         = {2017},

  month        = {10}

}

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