GDP-Biotin-Fucose

Protocols are guidelines. Parameters need to be optimized by end users.

Materials

Assay Buffer: 25 mM Tris, 10 mM MnCl₂, pH 7.5

Sample glycoprotein

Recombinant Fucosyltransferase

GDP-Biotin-Fucose (R&D Systems, Catalog # ES202)

Protein Sample Loading Dye

SDS-PAGE and Western Blot reagents or equivalent

TBST Buffer: 25 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.5

Commercial ECL (Enhanced Chemiluminescence) Reagents

Nitrocellulose Membrane

10% Fat Free Milk

Streptavidin-HRP (R&D Systems, Catalog # DY998)

Assay Procedure

1. Prepare reaction mixture by combining a sample glycoprotein (ideally between 1 to 10 µg), 0.25 nmol GDP-Biotin-Fucose, 0.5 μg of a Recombinant Fucosyltransferase, and add Assay Buffer to the final volume to 30 μL.
2. Prepare a negative control by repeating above but omitting the Fucosyltransferases.
3. Incubate all the reactions and controls at 37 °C for 60 minutes.
4. Stop the reactions and controls by adding appropriate volume of Protein Sample Loading Dye to each reaction.
5. Separate the reactions and controls by SDS-PAGE.
6. Blot the gel to a nitrocellulose membrane.
7. Block the blot with 10% fat-free milk for 5 minutes.
8. Thoroughly wash the membrane with TBST Buffer by changing buffer three times for a total of 30 minutes.
9. Incubate the blot with 25 ng/mL Streptavidin-HRP in 30 mL TBST buffer for 30 minutes.
10. Thoroughly wash the membrane with TBST Buffer by changing buffer three times for a total of 45 minutes.
11. Detect with Commercial ECL (Enhanced Chemiluminescence) Reagents.

Final Assay Conditions Per Reaction

GDP-Biotin-Fucose (R&D Systems®, Catalog # ES202): 0.25 nmol

Recombinant fucosyltransferase: 0.5 μg

Sample glycoproteins:  1-10 µg