Cy5-Fuc Labeled N2f

Catalog # Availability Size / Price Qty
GL304-100
Cy5-Fuc Labeled N2f
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Cy5-Fuc Labeled N2f Summary

 

Key Benefits

Learn more about Fluorescent Glycan Labeling and Detection

Formula

Fuc1Man3GlcNAc4Cy5

Molecular Weight

2292.16 Da

Formulation

Supplied in 20 mM Tris, pH 8.0

Stability & Storage

Store at < -20 °C. Good for 6 months from date of receipt.

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.
  • Store the unopened product at < -20 °C. Good for 6 months from date of receipt.

For Details:

 

Related Reagents
Labeled glycans

Click Chemistry

Enzymes and detection reagents

 

Specifications

Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Product Datasheets

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Scientific Data

Cy5-Fuc Labeled N2f

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).

Background: Glycan N2f

Long Name
Glycan N2f
Alternate Names
Glycan N2f

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

  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.
​        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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