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UDP-Azido-GalNAc

Activated Sugar

Key Product Details

UDP-Azido-GalNAc

Assay Procedure

Sample Protocol for Labeling Glycoprotein with O-glycan Replacement

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

 

Materials

Assay Procedure

1. Prepare a reaction mixture by combining 5 µg Protein Sample, with 1 µg rhGALNT2, 0.2 µg rE. faecalis O-Glycosidase, 0.1 µg rcpNeuraminidase, 0.5 nmol UDP-Azido-GlcNAc in the Assay Buffer with the final volume of 25 µL.

 

2. Prepare negative controls according to step 1 but omit rhGALNT2 or UDP-Azido-GlcNAc.

 

3. Incubate all the reactions and controls at 37°C for one hour.

 

4. 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.

 

5. 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 with commercial ECL (Enhanced Chemiluminescence) reagents.

Final Assay Conditions Per Reaction

  • UDP-Azido-GalNAc: 0.5 nmol
  • rhGALNT2: 1 µg
  • rcpNeuraminidase: 0.1 µg
  • rE. faecalis O-Glycosidase : 0.2 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

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