CMP-Cy3-Sialic Acid

Strategies for N- and O- glycan labeling with CMP-Cy3-Sialic Acid. Only representative glycans are depicted here. Glycans can be labeled with or without neuraminidase treatment. A selected neuraminidase will remove existing sialic acids and create more labeling sites for a selected sialyltransferase. While Cy5 conjugated sialic acid can be recognized by most of known sialyltransferases, it is poorly recognized by any known neuraminidase, which allows for labeling in a single reaction. The pairing of a neuraminidase and a sialyltransferase is based on the compatibility between the two enzymes. For example, Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM) can remove both α2-3 and α2-6 linked sialic acids, and it can be paired with all α2-3 specific sialyltransferases (ST3GALs) and all α2-6 specific sialyltransfeases (ST6GALs). In the case that α2-8 linked sialic acid needs to be labeled, specific neuraminidase such as Recombinant M.viridifaciens Neuraminidase Protein (Catalog # 5084-NM) and α2-8 specific sialyltransferases (ST8SIAs) will be paired.

Labeling bovine fetuin (Fet) using CMP-Cy3-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 TCE image (top panel) and a fluorescent imager (bottom 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. ST6GAL1 also showed some self-labeling in the presence of neuraminidase (indicted with an arrow in the bottom panel). M, Western blot molecular marker.

Labeling recombinant MUC16 using CMP-Cy3-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 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 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 (top panel) and a fluorescent imager (bottom panel). The bands of MUC16 corresponding to the neuraminidase treated samples appear to be darker than those of without neuraminidase treatment because of the incorporation of Cy3. M, Western blot molecular marker.