CMP-Azido-Sialic Acid
Catalog # ES102 | R&D Systems, Inc. a Bio-Techne Brand
Key Product Details
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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 Buffer: 25 mM HEPES, 150 mM NaCl, pH 7.5
- Fetuin (Sigma), 50 mg/mL stock in deionized water
- Recombinant Human ST3GAL1 (R&D Systems, Catalog # 6905-GT)
- Recombinant C. perfringens Neuraminidase (R&D Systems, Catalog # 5080-NM)
- CMP-Azido-Sialic Acid (R&D Systems, Catalog # ES102)
- Biotinylated Alkyne (R&D Systems, Catalog # ES100)
- MnCl2, 100 mM in deionized water
- 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 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
Citations for CMP-Azido-Sialic Acid (1)
Citations are publications that use Bio-Techne products. Selected citations for CMP-Azido-Sialic Acid include:
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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.