Lectin Blotting of Cell Lysates.

Abstract

Glycans are composed of branched structures consisting of monosaccharides, such as glucose and galactose linked by glycosidic bonds. Glycans are often bound to proteins and lipids and are localized at the cell surface. They are deeply involved in a wide range of multicellular systems inside and outside the cells, such as the quality control of glycoproteins, cell-cell communication, and various diseases. While western blotting uses antibodies to detect proteins, lectin blotting uses lectins, which are glycan-binding proteins, to detect glycans on glycoconjugates, such as glycoproteins. Lectin blotting was first reported in the early 1980s and has been widely used in life science for several decades. However, it is not straightforward to obtain consistent data using lectin blotting, which tends to show high backgrounds and lab-to-lab variation. Here, we describe the protocol used in our laboratory for lectin blotting following protein separation by SDS-PAGE to detect glycoproteins extracted from cell membrane fractions. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Extraction and quantification of proteins from cell lysate Basic Protocol 2: Lectin peroxidase labeling and lectin blotting.