These courses are being offered by the Complex Carbohydrate Research Center (CCRC) in the summer of 2013. Three of these courses are funded by the DOE-funded Plant and Microbial Carbohydrate Center, the NIH Integrated Technology Resource for Biomedical Glycomics and the NIH Integrated Technology Resource for Glycotechnology. The other course is funded by the NSF-funded grant supporting the generation, characterization and utilization of plant cell wall glycan-directed monoclonal antibodies.
This course will cover immunological techniques for studies of plant cell walls. Course participants will learn basic immunofluorescent labeling methods, as well as fixation, embedding and sectioning of plant tissues. We will also cover Enzyme-linked Immunosorbent Assays (ELISAs) as applied to plant cell wall extracts and polysaccharides, and cover Glycome Profiling as a rapid screen for plant cell wall composition. Participants will gain hands-on experience in each of these techniques, which will utilize the extensive plant cell wall glycan-directed monoclonal antibody collection generated at the CCRC. Participants may bring a sample of their own to analyze using these experimental approaches. Lectures will introduce these and other immunological approaches for plant cell wall studies. Experience with basic biochemical techniques is a prerequisite for participation.
This course will cover the techniques of glycosyl residue (composition by alditol acetate and methanolysis) and glycosyl linkage (methylation) analyses using gas-liquid chromatography/mass spectrometry, high pH Anion-exchange chromatography of oligosaccharides and monosaccharides and purification and isolation of polysaccharides. We will also cover lignin analysis by chemical methods and Pyrolysis Molecular beam mass spectrometry (Py-MBMS) and Pyrolysis GC-MS. Participants will also perform analyses of purified oligosaccharides, and they may bring one of their own samples. Lectures and demonstrations will cover other techniques for structural analysis. In addition, a choice of modules either on using mass spectrometry, computer modeling, or NMR spectroscopy for analysis of glycoconjuagtes will be included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation.
Participants will learn basic techniques for the isolation and characterization of oligosaccharides via a combination of hands-on work, lectures and demonstrations. Mixtures of oligosaccharides or glycopeptides derived from glycoproteins will be resolved into individual components by chromatographic procedures such as size-exclusion, HPLC and lectin affinity chromatography. Glycosphingolipids will be separated by thin-layer chromatography, and detection protocols will be discussed. The use of lectin blotting techniques for characterization of oligosaccharide structural features will be covered. Other topics to be included are release and structural characterization of O-linked glycans, and monosaccharide composition analysis. In addition, modules on using mass spectrometry, computer modeling or NMR spectroscopy for analysis of glycoconjugates will be included (see descriptions below). Experience with basic biochemical techniques is a prerequisite for participation.
On the Friday of each course, participants can choose one of the following three modules, Mass Spectrometric (MS) Analysis of Glycoconjugates, Computational Simulations of Carbohydrate Conformation and NMR of Carbohydrates. The course participants should indicate on the course application form which module they would be interested in taking.
Mass Spectrometric (MS) Analysis of Glycoconjugates:
The use of mass spectrometry for the characterization of glycoconjugates will be discussed. Topics in this area will include identifying occupied glycosylation sites by LC-MS and sequencing glycoprotein glycans with sequential exoglycosidase digestions followed by MS. Laboratory demonstrations will also be performed on these two topics.
Computational Simulations of Carbohydrate Conformation: The course will present a discussion of the conformational preferences of oligosaccharides. Focus will then shift to the prediction and validation of oligosaccharide structures produced computationally with molecular dynamics simulations with the AMBER/GLYCAM force field. NMR of Carbohydrates:
This module will be an introduction to the application of NMR spectroscopy of polysaccharides and glycoconjugates covering i) essential features of NMR spectra, ii) protocols used to solve primary structures, iii) applications and examples from the current literature, and iv) resources for information. Our four NMR instruments (300-, 500-, 600- and 800 MHz) will be used for demonstrations during this module.
** The course will feature hands-on laboratory work and will include demonstrations and lectures. A lab manual including selected analytical techniques and references will be provided. The cost of registration (including lunch) is $500 per course for individuals from nonprofit institutions; $1100 for others. The cost of travel and lodging is not included.
Participants will learn basic techniques for the structural characterization of glycosaminoglycans (GAGs), which will include depolymerization of the GAG polysaccharides to its disaccharide constituents using appropriate enzymes. The disaccharides will be separated by ion exchange chromatography and the products will be detected by U.V. The disaccharides will also be derivatized with 2-cyanoacetamide and will be detected with fluorescent detection. In a separate experiment, the glycosaminoglycans will be hydrolyzed by nitrous acid and the products will be analyzed. Mass spectrometry techniques for analysis of di- and oligosaccharides derived from glycosaminoglycans will also be covered. Experience with basic biochemical techniques is a prerequisite for participation
** The course will feature hands-on laboratory work and will include demonstrations and lectures. A lab manual including selected analytical techniques and references will be provided. The cost of registration (including lunch) is $600 per course for individuals from nonprofit institutions; $1400 for others. The cost of travel and lodging is not included.
For further information and an application form contact Dr. Parastoo Azadi, CCRC, 315 Riverbend Road, The University of Georgia, Athens,
For course personnel please click here.