GDP-Azido-Fucose
Catalog # ES101 | R&D Systems, Inc. a Bio-Techne Brand
Key Product Details
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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
Citations for GDP-Azido-Fucose (4)
Citations are publications that use Bio-Techne products. Selected citations for GDP-Azido-Fucose include:
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Species: Hamster
Sample Types: Whole Cells
Applications: BioassayF Pennarubia et al. (2020-08-28), Mouse WIF1 Is Only Modified with O-Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites Biomolecules, 2020-010(9).
PMID: 32872229 -
Species: E. coli
Sample Types: Whole Cells
Applications: BioassayM Deschuyter et al. (2020-05-31), Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer Cancers (Basel), 2020-012(6).
PMID: 32486426 -
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click ChemistryWu Z et al. (2018-02-01), Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-00(0).
PMID: 29186441 -
Sample Types: Recombinant Protein
Applications: BioassayWu Z et al. (2016-03-21), Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase. Biochem Biophys Res Commun, 2016-0473(2):524-9.
PMID: 27012207
There are no citations that match your criteria.