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CMP-Azido-Sialic Acid

Activated Sugar

Key Product Details

CMP-Azido-Sialic Acid

Assay Procedure

Sample Protocol for Labeling and Detecting O-glycan on Fetuin with ST3GAL1

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

 

Materials

Assay Procedure

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.

Final Assay Conditions Per Reaction

  • CMP-Azido-Sialic Acid: 0.5 nmol
  • rhST3GAL1: 1 µg
  • Desialylated Fetuin: 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

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