CMP-C9-Biotin-Sialic Acid

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 sialyltransferases

Recombinant neuraminidase

CMP-C9-Biotin-Sialic Acid (R&D Systems^®, Catalog # ES201)

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

SDS-PAGE and Western Blot reagents or equivalent

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

10% Fat Free Milk

Assay Procedure

1. Prepare reaction mixture by combining a sample glycoprotein (ideally between 1 to 10 µg), 0.5 µg of a recombinant sialyltransferase, 0.1 µg of recombinant neuraminidase, 1 nmol CMP-C9-Biotin-Sialic Acid, in the Assay Buffer with the final volume of 25 μL.
2. Prepare negative controls by combining a sample glycoprotein (ideally between 1 to 10 µg), 0.1 µg of recombinant neuraminidase, 1 nmol CMP-C9-Biotin-Sialic Acid in the Assay Buffer with the final volume of 25 μL.
3. Incubate reactions and controls at 37 °C for 30 minutes.
4. Separate the reactions and controls by SDS-PAGE.
5. Blot the gel to a nitrocellulose membrane.
6. Block the blot with 10% fat-free milk for 5 minutes.
7. Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 30 minutes.
8. Incubate the blot with 25 ng/mL Streptavidin-HRP in 30 mL TBST buffer for 30 minutes.
9. Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 45 minutes.
10. Detect with commercial ECL (Enhanced Chemiluminescence) reagents.

Final Assay Conditions Per Reaction

CMP-C9-Biotin-Sialic Acid :  1 nmol

Recombinant sialyltransferase:  0.5 µg

Recombinant neuraminidase : 0.1 µg

Sample glycoproteins:  1-10 µg