Generating Isoform-Specific Antibodies: Lessons from Nucleocytoplasmic Glycoprotein Skp1

Christopher M. West*, Hanke Van Der Wel, Zoiesha Chinoy, Geert Jan Boons, Ted J. Gauthier, Carol M. Taylor, Yuechi Xu

*Corresponding author for this work

    Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

    Abstract

    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1, 4(trans)- hydroxyproline linkage. Skp1 isoform-specific antibodies were successfully obtained by immunizing mice or rabbits with KLH-coupled synthetic peptides bearing either unmodified Pro, 4(trans)-hydroxyproline, or D-GlcNAcα1,4 (trans)-hydroxyproline, and screening with corresponding BSA-conjugates or by Western blotting toward a panel of Skp1 isoforms. Antibodies specific for Skp1 or HO-Skp1 were not found in exhaustive murine trials, yet monospecific polyclonal antibodies were readily achieved in rabbits without crossadsorption. In all cases, antibodies were specific at the protein but not the peptide level, which suggests that conformation comprises part of the basis for recognition and which should be considered when developing screening strategies.

    Original languageEnglish
    Title of host publicationGlycoscience: Biology and Medicine
    PublisherSpringer
    Pages927-934
    Number of pages8
    ISBN (Print)9784431548416, 9784431548409
    DOIs
    Publication statusPublished - 1 Jan 2015

    Keywords

    • Conformational epitope
    • Cytoplasmic glycosylation
    • Hydroxyproline
    • Isoform-specific antibody
    • Monoclonal antibody
    • Oxygen
    • Polyclonal antibody
    • Skp1
    • Synthetic glycopeptides
    • Ubiquitin ligase

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