• 6 months from date of receipt, -20 to -70 °C as supplied. • 3 months, -20 to -70 °C under sterile conditions after opening. • Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
Applications
Used as a ligand for lectins and to study glycan protein interaction.
Used as a substrate for various glycosidases and glycosyltransferases.
Used as a scaffold for the synthesis of various glycan epitopes such as sialyl Lewis X structures.
Key Features and Benefits:
Excitation at 649 nm and emission at 671 nm.
The fluorescent dye Cy5 is conjugated to the C6 position of the core-6 fucose.
Can be separated on 15%-17% SDS-PAGE and directly visualized as a single band under a fluorescent imager.
Linear response range for Cy5-labeled glycans can be from 10 fmol to 100 pmol, depending on the sensitivity of detection.
Stepwise Enzymatic synthesis of sialyl Lewis X (sLeX) epitope.
Slex is highlighted in the scheme and was synthesized from N2 using increasing amounts of Recombinant Human B4GalT1 Protein (3609-GT), Recombinant Human ST3GAL4 His-tag Protein (10496-GT), and Recombinant Human Fucosyltransferase 7/FUT7 Protein (6409-GT). For details, please refer to Wu, ZL. et al. (2020) Glycobiology, 30:970. https://doi.org/10.1093/glycob/cwaa030. The glycan short names are based on non-reducing end nomenclature (doi: https://doi.org/10.1101/2021.01.16.426965).
Preparation & Storage
Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Assay Procedure
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
Dilute rhB4GalT1, rhST3GAL4, and rhFUT7 to 50 ng/μL in the Assay Buffer to create an enzyme mix.
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.
Mix 10 μL of the enzyme mix and 10 μL of the reaction mix in a centrifuge tube.
Prepare a negative control by mixing 10 μL of reaction mix with 10 μL of Assay Buffer.
Incubate the reaction and control at 37 °C for 4 hours.
Stop the reactions and controls by adding 4 μL of 6X Gel Loading Dye to each tube.
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.
Image the gel using a fluorescent imager for 10 seconds.
Note: To visualize intermediate products enzymes may be added step-wise.
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
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