UDP-Azido-GalNAc
Catalog # ES103 | R&D Systems, Inc. a Bio-Techne Brand
Key Product Details
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Assay Procedure
Sample Protocol for Labeling Glycoprotein with O-glycan Replacement
Protocols are guidelines. Parameters need to be optimized by end users.
Materials
- Protein Sample
- Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MnCl2, pH 7.5.
- Recombinant Human GALNT2 (R&D Systems, Catalog # 7507-GT)
- Recombinant C. perfringens Neuraminidase (R&D Systems, Catalog # 5080-NM)
- Recombinant E. faecalis O-Glycosidase (R&D Systems, Catalog # 8886-GH)
- UDP-Azido-GalNAc (R&D Systems, Catalog # ES103)
- Biotinylated Alkyne (R&D Systems, Catalog # ES100)
- CuCl2, 1 mM in deionized water
- Ascorbic Acid, 20 mM in deionized water
- SDS-PAGE and Western blot reagents or equivalent
- TBST buffer: 25 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.5
- Streptavidin-HRP (R&D Systems, Catalog # DY998)
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
Citations for UDP-Azido-GalNAc (2)
Citations are publications that use Bio-Techne products. Selected citations for UDP-Azido-GalNAc include:
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Species: Human
Sample Types: Recombinant Protein
Applications: BioassayT Jaroentome et al. (2022-10-24), A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans Nature Communications, 2022-113(1):6325.
PMID: 36280670 -
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click ChemistryWu Z et al. (2018-02-01), Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-00(0).
PMID: 29186441
There are no citations that match your criteria.