UDP-Azido-GlcNAc

R&D Systems | Catalog # ES104

Activated Sugar
R&D Systems
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Key Product Details

Key Benefits

Learn more about Fluorescent Glycan Labeling and Detection

Species

Multi-Species

Conjugate

Azido

Product Summary for UDP-Azido-GlcNAc

 

UDP Azido GlcNAc Formula

Formula

C17H24N6O17P2

Molecular Weight

646.35 Da

Formulation

1 mM provided in 20 mM Tris, pH 8.0

Stability & Storage

Store the unopened product at < -20 °C. Good for 12 months from date of receipt.
 

Incorporate or detect GlcNAc without expensive, specialized equipment!

Applications

  • For in vitro enzymatic incorporation of azido-sugars into specific, targeted glycans.
  • Detecting the presence or absence of terminal GlcNAc residues (including O-GlcNAc).
  • Detecting T and Tn antigens.
  • Detecting the presence of high mannose glycan.

Key Features and Benefits

  • Can be introduced to proteins and lipids via various GlcNAc transferases.
  • Can be conjugated to desired reporter molecules via click chemistry.
  • Can be detected via Western blot, ELISA, and flow cytometry, depending on the type of reporter molecule.
  • Contains the smallest possible orthogonal functional group.
  • Has minimal side effects on target molecules.
  • User-friendly.

For Details: Wu et al., (2015) Carbohydrate Res. 412:1-6

Related Reagents

Click Chemistry

 

Enzymes and Detection Reagents for UDP-Azido-GlcNAc, ES104

GlcNAc Transferases

Specificity
Alpha-1,4-N-Acetylglucosaminyltransferase 4/A4GNT Adds GlcNAc to Galactose
Beta-1,3-N-Acetylglucosaminyltransferase 2/B3GNT2 Adds GlcNAc to Galactose
Beta-1,3-N-Acetylglucosaminyltransferase 4/B3GNT4 Adds GlcNAc to Galactose
Beta-1,3-N-Acetylglucosaminyltransferase 6/B3GNT6 Adds GlcNAc to GlaNAc
EOGT Adds O-GlcNAc to Ser/Thr (Extracellular)
Exostosin 1 Heparan sulfate synthesis
Exostosin 1/2 Heterodimer Heparan sulfate synthesis
Exostosin 2 Heparan sulfate synthesis
Exostosin-like 1/EXTL1 Heparan sulfate synthesis
Exostosin-like 2/EXTL2 Heparan sulfate synthesis
Exostosin-like 3/EXTL3 Heparan sulfate synthesis
Glucosaminyl (N-acetyl) Transferase 1/GCNT1 Adds GlcNAc to Galactose
N-Acetylglucosaminyltransferase I/MGAT1 Adds GlcNAc to mannose
N-Acetylglucosaminyltransferase III/MGAT3 Adds GlcNAc to mannose
N-Acetylglucosaminyltransferase V/MGAT5 Adds GlcNAc to mannose
O-GlcNAc Transferase/OGT Adds O-GlcNAc to Ser/Thr
POMGNT1 Part of MGAT family, adds GlcNAc to Ser/Thr-attached mannose

 

 

Schematic

Terminal GlcNAc
View Larger Image

Terminal GlcNAc can be detected on O- or N-Linked glycans. Glycans are removed by enzyme treatment. Specific transferases can be used to incorporate Azido-GlcNAc at suitable open positions. Azido-Sialic Acid can then be detected using Biotinylated Alkyne in a click chemistry reaction.

Sample Data

Assessing the Presence of open GlcNAc Incorporation Sites Using GlcNAc Transferases
View Larger Image

Assessing the Presence of open GlcNAc Incorporation Sites Using GlcNAc Transferases.
Total protein labeling is shown in the upper panel. Streptavidin-HRP detection is shown on the lower panel. UDP-Azido-GlcNAc is only incorporated into D-Fetuin (rcpNeuraminidase–treated Fetuin). The results indicate untreated Fetuin does not contain open sites for GlcNAc, whereas D-Fetuin contains incorporation sites that differ depending on enzyme specificity. In addition MW = molecular weight markers.

In each reaction, 10 µg Fetuin or D-fetuin sample, 0.3 nmol of UDP-Azido-GlcNAc, and 2 µg of the indicated GlcNAc transferases were mixed in 50 µL of 25 mM Tris supplemented with 10 mM of MnCl2 and 150 mM NaCl at pH 7.5. The reaction was incubated at 37 °C for a minimum of 20 minutes. The reactions were then conjugated with 0.1 mM Biotinylated Alkyne in the presence of 2 mM of Ascorbic Acid and 0.1 mM of CuCl2. The reactions were incubated at room temperature for 30 minutes. The reactions were then separated with 12% SDS-PAGE and blotted to a nitrocellulose paper and detected with Streptavidin-HRP.

Fetuin, from Sigma Aldrich, was further purified with Gel filtration column. Fetuin was treated with rcpNeuraminidase to prepare D-fetuin

Formulation, Preparation, and Storage

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

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 Documents for UDP-Azido-GlcNAc

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Download SDS

Product Specific Notices for UDP-Azido-GlcNAc

For research use only

Related Research Areas

Glycobiology-related Enzymes

Citations for UDP-Azido-GlcNAc

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Protocols

View specific protocols for UDP-Azido-GlcNAc (ES104):

Sample Protocol for GlcNAc Detection on Core-1 O-glycan

Protocols are guidelines. Parameters need to be optimized by end users.

 

Materials

Assay Procedure

1. Prepare a reaction mixture by combining 5 µg of Protein Sample, 1 µg of rhGCNT1 in the presence of 1 nmol of UDP-Azido-GlcNAc in the Assay Buffer with the final volume of 25 µL.

 

2. Prepare negative controls according to step 1 but omit Protein Sample or rhGCNT1.

 

3. Incubate all the reactions and the controls at 37°C for one hour.

 

4. To each of the samples, add 5 µL of 1 mM CuCl2, 5 µL of 20 mM Ascorbic Acid, and 5 µL of 1 mM Biotinylated Alkyne. Mix with gentle tapping.

 

5. Incubate all samples at room temperature for 1 hour.

 

6. Separate the reactions and controls by SDS-PAGE.

 

7. Blot the gel to a nitrocellulose membrane.

 

8. Block the blot with 10% fat-free milk for 5 minutes.

 

9. Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 45 minutes.

 

10. Incubate blot with 25 ng/mL Streptavidin-HRP in 30 mL TBST buffer for 30 minutes.

 

11. Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 45 minutes.

 

12. Detect with commercial ECL (Enhanced Chemiluminescence) reagents.

Final Assay Conditions Per Reaction

  • UDP-Azido-GlcNAc: 1 nmol
  • rhGCNT1: 1 µg
  • Protein Sample: 5 µg
  • Reaction Volume: 25 µl

Click Chemistry Reaction Conditions Per Reaction

  • CuCl2: 5 nmol
  • Ascorbic Acid: 100 nmol
  • Biotinylated Alkyne: 5 nmol
  • Reaction volume: 40 µl