GDP-Cy3-Fucose

Strategies for substrate glycan labeling with GDP-Cy3-Fucose. Only terminal lactosamine structure on N-glycans are depicted here for FUT6 or FUT9 labeling. Substrate glycans for other fucosyltransferases can be labeled under the same principle.

Labeling bovine fetuin (Fet) using GDP-Cy3-Fucose. Bovine fetuin was purified from crude fetuin by gel filtration. Samples of fetuin were labeled with Recombinant Human Fucosyltransferase 9/FUT9 Protein (Catalog # 9347-GT). In control lanes, no fetuin was present, revealing the self-labeling of FUT9 itself. Each lane contained 1 μg of fetuin except the controls. Samples were not labeled in the absence of neuraminidase but labeled strongly on asialo-fetuin (aFet) (generated by addition of Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM), Samples were separated on 4-20% gradient SDS-PAGE and imaged with TCE staining (top panel) and a fluorescent imager (lower panel). The bands of fetuin corresponding to the neuraminidase treated samples appear to be darker than those of without neuraminidase treatment because of the incorporation of Cy3.

Cellular glycan imaging with GDP-Cy3-Fucose. HeLa cells were stained by incorporation GDP-Cy3-Fucose (Catalog # ES401) by Recombinant Human Fucosyltransferase 9/FUT9 Protein (Catalog # 9347-GT) (green). Hela cells on a 96 well plate were briefly treated with Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM) to remove preexisting sialic acids. The treated cells were then labeled by FUT9 in the presence of GDP-Cy3-Fucose for 30 minutes. After the labeling, the solution was quickly removed by aspiration and washing two times with PBS. Afterwards, the cells were fixed and stained with DAPI (red) in the presence of 0.5% Triton X 100 to reveal the cell nuclei. These images show that N-glycans on HeLa cells display differential expression. For details on cell imaging with fluorophore-conjugated sugars, please refer to Wu. L. et al., (2020) Glycobiology 30:454-462.