UDP-Azido-GalNAc

Activated Sugar
Catalog # Availability Size / Price Qty
ES103-100
Product Details
Procedure
Citations (1)
FAQs
Supplemental Products
Reviews

UDP-Azido-GalNAc Summary

 

UDP Azido GalNAc Formula

Formula

C17H24N6O17P2

Molecular Weight

646.35 Da

Formulation

1 mM provided in 20 mM Tris, pH 8.0

Stability & Storage

Store the unopened product at < -20 °C. Good for 12 months from date of receipt.

 

Incorporate or detect GalNAc without expensive, specialized equipment!

Applications

  • For in vitro enzymatic incorporation of azido-sugars into specific, targeted glycans.
  • Detect the presence or absence of GalNAc modifications.
  • Monitoring O-glycans.

Key Features and Benefits

  • Can be introduced to proteins and lipids via various GalNAc transferases.
  • Can be conjugated to desired reporter molecules via click chemistry.
  • Can be detected via Western blot, ELISA, and flow cytometry, depending on the type of reporter molecule.
  • Contain the smallest possible orthogonal functional group.
  • Has minimal side effect on target molecules.
  • User-friendly.

For Details: Wu et al., (2015) Carbohydrate Res. 412:1-6

Related Reagents

Click Chemistry

 

Enzymes and Detection Reagents for UDP-Azido-GalNAc, ES103

GalNAc Transferases: All transfer GalNAc to Ser/Thr to initiate mucin-type glycosylation

GALNT2
GALNTL1
Polypeptide GalNAc Transferase 1/GALNT1
Polypeptide GalNAc Transferase 10/GALNT10
Polypeptide GalNAc Transferase 11/GALNT11
Polypeptide GalNAc Transferase 13/GALNT13
Polypeptide GalNAc Transferase 3/GALNT3
Polypeptide GalNAc Transferase 4/GALNT4
Polypeptide GalNAc Transferase 7/GALNT7

 

 

Schematic

Glycans are Removed by Enzyme Treatment
View Larger Image

Glycans are removed by enzyme treatment. Specific transferases can be used to incorporate Azido-GalNAc at suitable open positions. The incorporation Azido-GalNAc can then be detected using Biotinylated Alkyne in a click chemistry reaction.

Sample Data

Labeling Asialofetuin with GALNT2
View Larger Image

Labeling Asialofetuin with GALNT2.
Lane 1 and 2 shows UDP-Azido-GalNAc transferred to asialofetuin by rhGALNT2. Lane 3 was a negative control in the absence of UDP-Azido-GalNAc.

In each reaction, 5 µg of Asialofetuin (Sigma Aldrich), 0.5 nmol of UDP-Azido-GlcNAc, 1 µg rhGALNT2 and 1 µg of rE. faecalis O-Glycosidase was mixed in 50 µL of 25 mM HEPES supplemented with 10 mM of MnCl2 and 150 mM NaCl at pH 7.5. The reactions were incubated at 37°C for 60 minutes. The reactions were then conjugated, at room temperature for 30 minutes, with 1.0 nmol of Biotinylated Alkyne in the presence of 100 nmol of Ascorbic Acid and 5 nmol of CuCl2 for a final volume of 60 µL. The reactions were then separated with 12% SDS-PAGE and blotted to a nitrocellulose paper and detected with Streptavidin-HRP.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Multi-Species

Product Datasheets

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

Reagents Provided

Other Reagents Provided

Citation for UDP-Azido-GalNAc

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
    Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
    Glycobiology, 2017;0(0):.  2017

FAQs

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