CMP-Azido-Sialic Acid

1. Prepare Desialylated Fetuin by mixing Fetuin with rcpNeuraminidase at mass ratio of 100:1 in the Assay Buffer with the concentration of Fetuin at 1 mg/mL for 20 minutes at room temperature. The rcpNeuraminidase is then heat inactivated at 90°C for 5 minutes.

2. Prepare reaction mixture by combining 5 µg Desialylated Fetuin, 1 µg rhST3GAL1, 0.5 nmol CMP-Azido-Sialic Acid, in the Assay Buffer supplemented with 10 mM MnCl₂ with the final volume of 25 µL.

3. Prepare negative controls according to step 2 but omit rhST3GAL1 or CMP-Azido-Sialic Acid.

4. Incubate reactions and controls at 37 °C for 30 minutes.

5. 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. Incubate all samples at room temperature for 1 hour.

6. Separate the reactions and controls by 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 Detect with commercial ECL (Enhanced Chemiluminescence) reagents.