Cy5-Fuc Labeled N2f

Sample Assay Protocol 

One-pot Synthesis of Sialyl-Lewis X N-glycan using Cy5-Fuc Labeled N2f/Cy5-N2f as a scaffold

Suggested input of a Cy5-N2f in an assay separated on SDS-PAGE is from 0.01-1 pmol. To monitor the glycan intermediates, it is suggested that the enzymes are added stepwise. 

Protocols are guidelines. Parameters need to be optimized by end users.
Please refer to Wu, ZL. et al. (2020) Glycobiology, 30:970. https://doi.org/10.1093/glycob/cwaa030

Materials

• Assay Buffer: 25 mM Tris, 10 mM MnCl₂, pH 7.5
• Recombinant Human ST3GAL4 His-tag Protein, CF (rhST3GAL4) (Catalog # 10496-GT) or Recombinant Human ST3GAL6 Fc Chimera Protein, CF (rhST3GAL6) (Catalog # 10591-GT)
• Recombinant Human B4GalT1 Protein, CF (rhB4GalT1) (Catalog # 3609-GT)
• Recombinant Human Fucosyltransferase 7/FUT7 Protein, CF (rhFUT7) (Catalog # 6409-GT)
• Cy5-Fuc Labeled N2f (Cy5-N2f) (Catalog # GL304)
• UDP-Gal
• CMP-NANA
• GDP-Fucose
• 15% SDS-PAGE
• 6X Gel Loading Dye
• A fluorescent imager

Assay

1. Dilute rhB4GalT1, rhST3GAL4, and rhFUT7 to 50 ng/μL in the Assay Buffer to create an enzyme mix.
2. Create a reaction mix by combining 0.02 µM Cy5-N2f and 1 mM UDP-Gal, 1 mM CMP-NANA, and 1 mM GDP-Fucose in Assay Buffer.
3. Mix 10 μL of the enzyme mix and 10 μL of the 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 4 hours.
6. Stop the reactions and controls by adding 4 μL of 6X Gel Loading Dye to each tube.
7. Load 12 μL 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 for 10 seconds.

Final Assay Conditions Per Reaction

•  rhB4GalT1: 500 ng 
•  rhST3GAL4: 500 ng
•  rhFUT7: 500 ng
•  Cy5-N2f: 0.2 pmol
•  CMP-NANA: 0.5 mM
•  UDP-Gal: 0.5 mM
•  GDP-Fucose: 0.5 mM