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  • An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography

    Author(s)
    Lee, Ling
    Hincapie, Marina
    Packer, Nicolle H.
    Baker, Mark S.
    Hancock, William S.
    Fanayan, Susan
    Griffith University Author(s)
    Packer, Nicki
    Year published
    2012
    Metadata
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    Abstract
    Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. ...
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    Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.
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    Journal Title
    Journal of Separation Science
    Volume
    35
    Issue
    18
    DOI
    https://doi.org/10.1002/jssc.201200049
    Subject
    Analytical chemistry
    Analytical chemistry not elsewhere classified
    Other chemical sciences
    Biochemistry and cell biology
    Publication URI
    http://hdl.handle.net/10072/336565
    Collection
    • Journal articles

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