Submitted to: Annual Missouri Symposium
Publication Type: Abstract only
Publication Acceptance Date: 4/22/2005
Publication Date: 4/22/2005
Citation: Johnston, M.L., Miernyk, J.A. 2005. Use of immobilized-lectin chromatography to prepare samples for proteomic analysis [abstract]. Twenty-Second Annual Missouri Symposium. p. 80. Interpretive Summary:
Technical Abstract: The lectin Concanavalin A (Con A) specifically binds high-mannose-type glycans. Immobilized Con A is commonly used both for the purification of glycoproteins and the removal of glycoprotein "contaminants" during purification of non-glycosylated proteins. Proteomic analyses of developing soybean (Glycine max cv. Jack) seeds were complicated by the abundance of the N-glycosylated storage proteins. "Total" proteins were isolated from developing seeds by a commonly used method (Hurkman WJ, Tanaka CK (1986) Plant Physiol 81:802-806). After phenol extraction and precipitation with methanolic ammonium acetate, the protein pellets were dissolved in 8 M urea, 10% glycerol, and 0.5% Triton X-100. Con A-Sepharose was equilibrated in binding buffer (4 M urea, 20 mM HEPES-NaOH, pH 7.5, 2 mM MnCl2, and 2 mM CaCl2). The protein samples were diluted to 4 M urea by addition of binding buffer minus urea. Analysis of the protein fraction that did not bind to the lectin, by SDS-PAGE, revealed a substantial band at the position corresponding to the Con A subunit (Mr 26,000). All of the denaturing buffers tested resulted in "bleeding" of Con A subunits from the affinity matrix. This problem was solved by chemically cross-linking the Con A subunits. The immobilized lectin was equilibrated in PBS, pH 8.5, then the subunits were cross-linked using 10 mM dimethlypalimidate plus 10 mM dimethylsuberimidate in PBS, pH 8.5. After 45 minutes, the reaction was stopped with 500 mM Tris-HCl, pH 8.8. Binding to cross-linked Con A-Sepharose effectively removed the glycinin storage proteins and other less abundant glycoproteins, without any bleeding of the Con A subunits, allowing a greater amount of sample protein to be analyzed on 2-D gels.