Jump to main content
Jump to site search

Issue 12, 2011
Previous Article Next Article

Fluorescent labeling agents change binding profiles of glycan-binding proteins

Author affiliations

Abstract

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin–glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of proteinligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein–glycan interactions.

Graphical abstract: Fluorescent labeling agents change binding profiles of glycan-binding proteins

Back to tab navigation

Supplementary files

Publication details

The article was received on 10 Aug 2011, accepted on 20 Sep 2011 and first published on 18 Oct 2011


Article type: Paper
DOI: 10.1039/C1MB05332A
Citation: Mol. BioSyst., 2011,7, 3343-3352
  •   Request permissions

    Fluorescent labeling agents change binding profiles of glycan-binding proteins

    Y. Fei, Y. Sun, Y. Li, K. Lau, H. Yu, H. A. Chokhawala, S. Huang, J. P. Landry, X. Chen and X. Zhu, Mol. BioSyst., 2011, 7, 3343
    DOI: 10.1039/C1MB05332A

Search articles by author

Spotlight

Advertisements