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Frontal affinity chromatography: sugar–protein interactions

Nature Protocols volume 2, pages 2529–2537 (2007)Cite this article

Abstract

Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar–protein interactions is ∼10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.

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Figure 1: Principle of frontal affinity chromatography.
Figure 2: Automated frontal affinity chromatography (FAC) system.
Figure 3
Figure 4: Preparation of a column.
Figure 5: Screening and analysis of Griffonia simplicifolia lectin-II (GSL-II).
Figure 6

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Acknowledgements

The authors thank Junko Kominami (J-Oil Mills), Noboru Uchiyama, Yusuke Osaka (Shimadzu Co.) and all current and past members of the Hirabayashi Lab for their various contributions to the development and establishment of the FAC protocol; J-Oil Mills for providing GSL-II. This work was supported by New Energy and Industrial Technology Development Organization (NEDO) in Japan under the Ministry of Economy, Trade, and Industry (METI).

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Authors and Affiliations

  1. Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Ibaraki, 305-8568, Japan

    Hiroaki Tateno, Sachiko Nakamura-Tsuruta & Jun Hirabayashi

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  1. Hiroaki Tateno
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Correspondence to Jun Hirabayashi.

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Screening and analysis of GSL-II (PDF 3789 kb)

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Tateno, H., Nakamura-Tsuruta, S. & Hirabayashi, J. Frontal affinity chromatography: sugar–protein interactions. Nat Protoc 2, 2529–2537 (2007). https://doi.org/10.1038/nprot.2007.357

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