title: Chemical cross-linking immobilized concanavalin A for use in proteomic analyses.
abstract: Lectin affinity chromatography was used to reduce the amount of the abundant glycoprotein beta-conglycinin in total protein samples prepared from developing soybean (Glycine max L. Merrill cv. Jack) seeds. Electrophoretic analysis of both the concanavalin A-Sepharose binding and non-binding fraction revealed an abundant protein band at Mr 26,000. The amount of this protein was greatly increased when concanavalin A-Sepharose was used with urea-containing buffers. Peptide mass fingerprint analysis of this abundant protein band unequivocally identified it as concanavalin A (con A). A simple and gentle method was used to chemically cross-link the con A subunits so that the lectin-Sepharose retained the ability to bind high-mannose type glycoproteins. The chemically cross-linked con A-Sepharose was stable in buffers that contained up to 8M urea, making this an affinity matrix suitable for use in electrophoresis-based proteomic analyses.
authors.affiliation: USDA, Agricultural Research Service, Plant Genetics Research Unit, University of Missouri, Columbia, Missouri 65211, USA. miernykj@missouri.edu
authors.forename: Jan A
authors.initials: JA
authors.lastname: Miernyk
authors.forename: Mark L
authors.initials: ML
authors.lastname: Johnston
mesh_terms: D000595:Amino Acid Sequence; D003208:Concanavalin A / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012639:Seeds / Q000737:chemistry; D013025:Glycine max / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D010940:Plant Proteins; D003208:Concanavalin A