CMP-Cy5-Sialic Acid

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Labeling bovine fetuin (Fet) using CMP-Cy5-Sialic Acid. Bovine fetuin was purified from crude fetuin by gel filtration. Samples of fetuin were labeled through O-glycans with Recombinant Human ST3GAL1 Protein (Catalog # 6905-GT) or N-glycans with Recombinant Human ST6GAL1 (aa 44-406) Protein (Catalog # 7620-GT). Control lanes contain all components but lack a labeling enzyme. Each lane contained 1 μg of fetuin except the controls. The control lanes contained 2 μg of protein. Samples exhibited greater labeling in the presence of Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM) (Neu, indicated with + and - signs), suggesting that both N- and O-glycans on fetuin were largely sialylated before labeling. Samples were separated on 4-20% gradient SDS-PAGE and imaged with silver staining (left panel) and a fluorescent imager (middle and right panels at different contrasts). The bands of fetuin corresponding to the neuraminidase treated samples appear to be darker than those without neuraminidase treatment because of the incorporation of Cy5. ST6GAL1 also showed some self-labeling in the presence of neuraminidase (indicted with an arrow in the right panel). M, Western Blot molecular marker.

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Labeling recombinant MUC16 using CMP-Cy5-Sialic Acid. Samples were labeled through O-glycans with Recombinant Human ST3GAL1 Protein (Catalog # 6905-GT) or N-glycans with Recombinant Human ST6GAL1 (aa 44-406) Protein (Catalog # 7620-GT). Control lanes contain all components but lack a labeling enzyme. Each experimental lane contained 1 μg of Recombinant Human CA125/MUC16 Protein (Catalog # 5609-MU), while the control lanes contained 2 μg of protein. Samples exhibited greater labeling in the presence of Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM) (Neu, indicated with + and - signs), suggesting that both N- and O-glycans on MUC16 were largely sialylated before labeling. In fact, by densitometry analysis on the fluorescent bands of MUC16 before and after neuraminidase treatment, the sialyation of the protein was measured to be 91.3% for O-glycans and 79.9 % for N-glycans. Samples were separated on 4-20% gradient SDS-PAGE and imaged with TCE Image (left panel) and a fluorescent imager (middle and right panels at different contrasts). The bands of MUC16 corresponding to the neuraminidase treated samples appear to be more heavily labeled than those without neuraminidase treatment because of the incorporation of Cy5. ST6GAL1 also showed some self-labeling in the presence of neuraminidase (indicted with an arrow in the middle panel). M, Western Blot molecular marker.

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Cellular glycan imaging of Cy5-Sialic Acid and CMP-Alexa Fluor® 488-Sialic Acid. The O- and N-glycans on HeLa cells were stained by incorporation of Cy5-Sialic Acid by Recombinant Human ST3GAL1 Protein (Catalog # 6905-GT) (red) and Alexa Fluor^® 555 by Recombinant Human ST6GAL1 (aa 44-406) Protein (Catalog # 7620-GT) (green). HeLa cells on a 96-well plate were briefly treated with Recombinant M.viridifaciens Neuraminidase (Catalog # 5084-NM) to remove preexisting sialic acids. The treated cells were then labeled on O-glycans by ST3GAL1 in the presence of CMP-Cy5-Sialic Acid (Catalog # ES302) for 20 minutes. After the labeling, the solution was quickly removed by aspiration followed by washing two times with PBS. The cells were then subjected to the second labeling on N-glycans by ST6GAL1 in the presence of CMP-Alexa Fluor^® 488-Sialic Acid (green). Afterwards, the cells were fixed and stained with DAPI (blue) in the presence of 0.5% Triton X 100 to reveal the cell nuclei. All four panels were from the same viewing area and imaged under different channels. These images show that O-glycans on HeLa cells display differential expression. For details of cell glycan imaging, please visit our paper in press: Wu. L. et al., (2020) Glycobiology 30:454-462.