Learn more about Fluorescent Glycan Labeling and Detection
1 mM Provided in 20 mM Tris, 50 mM NaBorate, pH 9.0
Incorporate or detect fucose without expensive, specialized equipment!
- For in vitro enzymatic incorporation of azido-sugars into specific, targeted glycans.
- Detecting the presence or absence of fucosylated glycans.
- Tagging bio-molecules through fucosylation.
- Assessing the level of fucosylation on target molecules.
Key Features and Benefits
- Can assess for the presence of terminal fucosylation (e.g. core fucose) without expensive, specialized equipment.
- Can be introduced to proteins and lipids that have fucosylation sites via fucosyltransferases.
- 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.
Wu, ZL et al., (2015) Carbohydrate Res. 412:1-6.
Wu, ZL. et al., (2020) Glycobiology 30:970.
Enzymes and Detection Reagents for GDP-Azido-Fucose, ES101
|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|
|Generates sLex antigen|
|Fucosyltransferase 7/FUT7||Adds (terminal) Fucose to GlcNAc||Generates sLex antigen|
|Fucosyltransferase 8/FUT8||Adds (non-terminal) Fucose to GlcNAc|
|Generates sLex antigen|
|Fucosyltransferase 11/FUT11||Adds (non-terminal) Fucose to GlcNAc|
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.
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.
Sample Protocol for Core-6 Fucose Labeling
Protocols are guidelines. Parameters need to be optimized by end users.
- 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)
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
Citations for GDP-Azido-Fucose
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 4
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Mouse WIF1 Is Only Modified with O-Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites
Authors: F Pennarubia, E Pinault, B Al Jaam, CE Brun, A Maftah, A Germot, S Legardinie
Biomolecules, 2020-08-28;10(9):. 2020-08-28
Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer
Authors: M Deschuyter, F Pennarubia, E Pinault, S Legardinie, A Maftah
Cancers (Basel), 2020-05-31;12(6):. 2020-05-31
Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
Glycobiology, 2018-02-01;0(0):. 2018-02-01
Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.
Authors: Wu Z, Zhou H, Ethen C, N Reinhold V
Biochem Biophys Res Commun, 2016-03-21;473(2):524-9. 2016-03-21
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