Cy5-Fuc Labeled M1N1f

R&D Systems | Catalog # GL301

R&D Systems
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Key Product Details

Key Benefits

Learn more about Fluorescent Glycan Labeling and Detection

Conjugate

Cy5

Product Summary for Cy5-Fuc Labeled M1N1f

Cy5 Labeled M1N1f
 

Formula

Fuc1Man3GlcNAc3Cy5

Molecular Weight

2088.96 Da

Formulation

Supplied in 20 mM Tris, pH 8.0

Stability & Storage

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

For Details:

Related Reagents

Click Chemistry

Enzymes and detection reagents

 

Data Examples

Cy5 Labeled M1N1f as a substrate for recombinant MGAT2 assay.
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Cy5-Fuc Labeled M1N1f as a substrate for recombinant MGAT2 assay. Cy5-Fuc labeled M1N1f (M1N1f) (Catalog # GL301) (0.2 pmol per reaction) was treated by a serial dilution of Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 Protein (Catalog # 10711-GT). The reactions were incubated 30 minutes at 37oC then heat inactivated, and further elongated with Recombinant Human B4GalT1 Protein (Catalog # 3609-GT)  at 37oC for 20 minutes.  Samples were separated on 15% SDS-PAGE and imaged with TCE staining (top panel) and fluorescent imaging (lower panel). Product 2 is visible with 6.25 ng of MGAT2, and its maximal level was achieved at 100 ng of the enzyme. The conversion of M1N1f to M2f by MGAT2 did not generate obvious mobility shift (data not shown). To overcome this issue, the substrate M1N1f and the product M2f were elongated by B4GalT1 to Product 1 and Product 2, respectively, to  create an obvious gel mobility shift.

Scientific Data Images for Cy5-Fuc Labeled M1N1f

Cy5-Fuc Labeled M1N1f'

This substrate can be used as ligand for lectins and to study various glycosidases and glycosyltransferases. This can also be used as a scaffold for the synthesis of various glycan epitopes such as sialyl Lewis X structures. The fluorescent dye Cy5 is conjugated to the C6 position of the core-6 fucose.

Formulation, Preparation, and Storage

Formulation

No Formulation Info in Bulk Inventory

Reconstitution Instructions

Reconstitute at 100 μg/mL in PBS containing at least 0.1% human or bovine serum albumin.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

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.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: M1N1f

Long Name

M1N1f

Additional M1N1f Products

Product Documents for Cy5-Fuc Labeled M1N1f

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Cy5-Fuc Labeled M1N1f

For research use only

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Protocols

View specific protocols for Cy5-Fuc Labeled M1N1f (GL301):

Sample Assay Protocol for using Cy5-Fuc Labeled M1N1f/M1N1f as substrate for Recombinant Human N-acetylglucosaminyltransferase 2/MGAT2 assay

Protocols are guidelines. Parameters need to be optimized by end users. Suggested input of a Cy5-M1N1f in an assay separated on SDS-PAGE is from 0.01-1 pmol.

Materials

  • Assay Buffer: 25 mM Tris, 10 mM CaCl2, pH 7.5
  • Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 (rhMGAT2) (Catalog # 10711-GT)
  • Cy5-Fuc Labeled M1N1f (M1N1f) (Catalog # GL301)
  • Recombinant Human B4GALT1 (rhB4GALT1) (Catalog # 3609-GT)
  • UDP-Gal
  • UDP-GlcNAc
  • 15% SDS-PAGE
  • 6X gel loading dye
  • A fluorescent imager

Assay

  1. Dilute rhMGAT2 to 10 ng/µL in the Assay Buffer.
  2. Create a Reaction Mix #1 by combining 0.02 µM M1N1f and 1.5 mM UDP-GlcNAc in Assay Buffer.
  3. Mix 10 µL of the diluted rhMGAT2 and 10 µL of the Reaction Mix #1 in a centrifuge tube. 
  4. Prepare a negative control by mixing 10 µL of Assay Buffer and 10 µL of Reaction Mix #1 in another centrifuge tube.
  5. Incubate the reaction and control at 37o C for 30 minutes.
  6. Heat tubes to 90o C for 2 minutes, cool on ice, and quickly spin down condensation.
  7. Create Reaction Mix #2 by combining 50 ng/µL rhB4GALT1 and 1.5 mM UDP-Gal in Assay Buffer.
  8. Add 10 µL of Reaction Mix #2 to each tube.
  9. Incubate at 37o C for 20 minutes.
  10. Stop the reactions and controls by adding 6 μL of 6X gel loading dye to each tube.
  11. Load 18 µL each of the above reactions and controls per well on a 15% SDS-PAGE and run down 80% length of the gel.
  12. Visualize the gel using a fluorescent imager for 10 seconds.

Final Assay Conditions Per Reaction

  • rhMGAT2: 100 ng 
  • Cy5-M1N1f: 0.2 pmol 
  • UDP-GlcNAc: 0.5 mM
  • rhB4GALT1: 500 ng     
  • UDP-Gal: 0.5 mM