Cy5-Fuc Labeled N2nf (Cy5-N2nf)

Sample Assay Protocol for testing activity of Glycosyltransferases using Cy5-Fuc Labeled N2nf/Cy5-N2nf as a substrate.

Suggested input of Cy5-N2nf in an assay separated on SDS-PAGE is from 0.01-1 pmol.

Protocols are guidelines. Parameters need to be optimized by end users, enzyme input and reaction time may need to be adjusted to accommodate specific activity of the enzyme.

Refer to Wu, ZL. et al. (2020) Glycobiology, 30:970. 
https://academic.oup.com/glycob/article/30/12/970/5815178
 

Materials

• Assay Buffer: 25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂,  pH 7.5 (dependent on requirements of Glycosyltransferase)
• Glycosyltransferase
• Appropriate Nucleotide Sugar (e.g. CMP-Sialic Acid, UDP-GlcNAc, GDP-Fucose, or UDP-Gal)
• 15% SDS-PAGE
• 6X Gel Loading Dye
• Fluorescent imager

Assay

1. Dilute Glycosyltransferase 10 to 100 ng/µL in the Assay Buffer.
2. Create a reaction mix by combining 0.02 µM Cy5-N2nf and 1 mM nucleotide sugar in Assay Buffer.
3. Mix 10 µL dilute Glycosyltransferase and 10 µL of reaction mix in a centrifuge tube.
4. Prepare a negative control by mixing 10 µL of reaction mix with 10 µL of Assay Buffer.
5. Incubate the reaction and control at 37 °C for 1 hour.
6. Stop the reactions and controls by adding 4 µL of 6X Gel Loading Dye to each tube.
7. Load 12 µL of each of the above reactions and controls per well on a 15% SDS-PAGE and run down 80% length of the gel.
8. Image the gel using a fluorescent imager using the red channel for 10 seconds.

Final Assay Conditions Per Reaction

• Glycosyltransferase: 1 to 10 µg 
• Cy5-N2nf: 0.2 pmol
• Nucleotide Sugar: 0.5 mM