GDP-Azido-Fucose
R&D Systems, part of Bio-Techne | Catalog # ES101
Key Product Details
| Key Benefits: |
Learn more about Fluorescent Glycan Labeling and Detection Summary for GDP-Azido-Fucose
Incorporate or detect fucose without expensive, specialized equipment! Applications
Key Features and Benefits
For Details: |
| Fucosyltransferases | Fucosylation | Notes |
| Protein O-Fucosyltransferase 1/POFUT1 | Adds Fucose to Ser/Thr | Important for Notch signaling |
| Fucosyltransferase 2/FUT2 | Adds (terminal) Fucose to Galactose | Generates H antigen |
| Fucosyltransferase 3/FUT3 | Adds (terminal) Fucose to GlcNAc | Generates Lea, Leb antigens |
| Fucosyltransferase 5/FUT5 | Adds (terminal) Fucose to Galactose | Generates sLex antigen |
| Fucosyltransferase 6/FUT6 | Generates sLex antigen | |
| Fucosyltransferase 7/FUT7 | Adds (terminal) Fucose to GlcNAc | Generates sLex antigen |
| Fucosyltransferase 8/FUT8 | Adds (non-terminal) Fucose to GlcNAc | |
| Fucosyltransferase 9/FUT9 | Generates sLex antigen | |
| Fucosyltransferase 11/FUT11 | Adds (non-terminal) Fucose to GlcNAc |
Schematic
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| The terminal fucose can be removed using a Fucosidase. Azido-fucose can then be added to open sites with specific fucosyltransferases and detected using Biotinylated Alkyne in a click chemistry reaction. |
Sample Data
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| Detecting Open Fucosylation Sites on the Acceptor Substrate Recombinant Drosophila Notch EGF20. rhPOFUT1 at 0.5 µg of was incubated at 37 °C for one hour with increasing amounts (0.5, 1, 2, 4 µg) of rdNotch EGF20 repeat. The left side is a labeling reaction including GDP-Azido-Fucose. The right side serves as a negative control labeling reaction without GDP-Azido-Fucose. The results clearly show GDP-Azido-Fucose incorporation into the Notch EGF20 substrate on open sites for POFUT1-mediated fucosylation. The buffer used contained 25 mM HEPES pH 7.5, 150 mM NaCl, and 10 mM MnCl2 for a total volume of 40 µl. The reactions were then conjugated for one hour with 1 nmol of Biotinylated Alkyne in the prescence of 100 µM CuCl2 and 2 mM Ascorbic Acid for a final volume of 50 µl. All reactions were separated on 12% SDS-PAGE and then detected with Streptavidin-HRP. |
Assay Procedure
Sample Protocol for Core-6 Fucose Labeling
Protocols are guidelines. Parameters need to be optimized by end users.
Materials
- Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MnCl2, pH 7.5.
- Protein Sample
- Recombinant Human Fucosyltransferase 8/FUT8 (R&D Systems, Catalog # 5768-GT)
- GDP-Azido-Fucose (R&D Systems, Catalog # ES101)
- 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 rhFUT8 in the presence of 1 nmol GDP-Azido-Fucose in the Assay Buffer with the final volume of 25 µL.
2. Prepare negative controls according to step 1 but omit Protein Sample or rhFUT8 or GDP-Azido-Fucose.
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 12% 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 the 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
- GDP-Azido-Fucose: 1 nmol
- rhFUT8: 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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