GDP-Azido-Fucose

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. Add to each of the samples: 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