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Immunology JoVE (Open Access) Citable · DOI

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

DOI: 10.3791/56424-v
What you'll learn
  • ✓ Prepare plant cell wall polysaccharides for structural analysis via PACE
  • ✓ Perform enzymatic hydrolysis and derivatization of oligosaccharides
  • ✓ Interpret polyacrylamide gel electrophoresis results for mannan characterization
Protocol

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Difficulty
advanced
Total time
~3–4 hours per sample (hydrolysis overnight; gel running ~2 hrs)
Model organism
Arabidopsis thaliana (plant tissue)
Biosafety
BSL-1

Steps

1
Aliquot and pre-treat alcohol-insoluble residue

Divide plant cell wall material (AIR) into working portions and perform pre-treatment to remove interfering compounds and prepare for enzymatic digestion.

▶ 00:37
2
Hydrolyze AIR with glycosyl hydrolases

Incubate pretreated AIR with specific hydrolase enzymes to break down mannan and other polysaccharides into oligosaccharide fragments.

▶ 02:11
3
Derivatize oligosaccharide products

Apply fluorescent or chromatic labeling to hydrolyzed oligosaccharides to enable detection during gel electrophoresis.

▶ 03:20
4
Prepare polyacrylamide PACE gels

Cast and polymerize polyacrylamide gels with appropriate buffer and acrylamide concentration for separating oligosaccharides by size and charge.

▶ 04:29
5
Run samples on PACE gel

Load derivatized oligosaccharide samples onto gel and apply electrical field to separate compounds based on molecular weight and charge.

▶ 06:33
6
Visualize and document gel results

Image the gel using appropriate detection method (UV, fluorescence, or staining) to visualize separated oligosaccharide bands.

▶ 07:51
7
Interpret mannan structure characterization

Analyze band patterns and mobility to determine mannan composition, linkage types, and structural features of plant cell wall polysaccharides.

▶ 09:07
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