UDP-Azido-GlcNAc

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Terminal GlcNAc can be detected on O- or N-Linked glycans. Glycans are removed by enzyme treatment. Specific transferases can be used to incorporate Azido-GlcNAc at suitable open positions. Azido-Sialic Acid can then be detected using Biotinylated Alkyne in a click chemistry reaction.

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Assessing the Presence of open GlcNAc Incorporation Sites Using GlcNAc Transferases.
Total protein labeling is shown in the upper panel. Streptavidin-HRP detection is shown on the lower panel. UDP-Azido-GlcNAc is only incorporated into D-Fetuin (rcpNeuraminidase–treated Fetuin). The results indicate untreated Fetuin does not contain open sites for GlcNAc, whereas D-Fetuin contains incorporation sites that differ depending on enzyme specificity. In addition MW = molecular weight markers.

In each reaction, 10 µg Fetuin or D-fetuin sample, 0.3 nmol of UDP-Azido-GlcNAc, and 2 µg of the indicated GlcNAc transferases were mixed in 50 µL of 25 mM Tris supplemented with 10 mM of MnCl₂ and 150 mM NaCl at pH 7.5. The reaction was incubated at 37 °C for a minimum of 20 minutes. The reactions were then conjugated with 0.1 mM Biotinylated Alkyne in the presence of 2 mM of Ascorbic Acid and 0.1 mM of CuCl₂. The reactions were incubated at room temperature for 30 minutes. The reactions were then separated with 12% SDS-PAGE and blotted to a nitrocellulose paper and detected with Streptavidin-HRP.

Fetuin, from Sigma Aldrich, was further purified with Gel filtration column. Fetuin was treated with rcpNeuraminidase to prepare D-fetuin