UDP-Azido-GlcNAc
R&D Systems, part of Bio-Techne | Catalog # ES104
Key Product Details
| Key Benefits: |
Learn more about Fluorescent Glycan Labeling and Detection |
Summary for UDP-Azido-GlcNAc
| 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 |
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 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. 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 |
Assay Procedure
Sample Protocol for GlcNAc Detection on Core-1 O-glycan
Protocols are guidelines. Parameters need to be optimized by end users.
Materials
- Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl2, pH 7.5.
- Protein Sample
- Recombinant Human GCNT1 (R&D Systems, Catalog # 7248-GT)
- UDP-Azido-GlcNAc (R&D Systems, Catalog # ES104)
- Biotinylated Alkyne (R&D Systems, Catalog # ES100)
- CuCl2, 1 mM in deionized water
- Ascorbic Acid, 20 mM in deionized water
- SDS-PAGE and Western blot reagents or equivalent
- TBST buffer: 25 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.5
- Streptavidin-HRP (R&D Systems, Catalog # DY998)
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
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