CMP-C9-Biotin-Sialic Acid Summary
Key Benefits
Learn more about Fluorescent Glycan Labeling and Detection
|
Formula |
C41H65N10O21PS |
|
Molecular Weight |
1097.05 Da |
|
Formulation |
Lyophilized with Tris, pH 8.0 |
|
Stability & Storage |
Store the unopened product at < -20 °C. Good for 12 months from date of receipt. |
Applications
- Allow direct biotinylation of sialoglycans of proteins and lipids.
- Detecting specific sialoglycans on glycoproteins.
- Detecting and imaging sialoglycans on cells and tissue samples.
Key Features and Benefits:
- Can be introduced to sialoglycans and glycolipids via various sialyltransferases.
- Allows glycan imaging on live cells.
- No side effects on target molecules observed.
- Convenient and user-friendly.
For Details:
Wu et al. (2015) Carbohydrate Res. 412:1-6.
Wu et al. (2019) Glycobiology, 29:750-754.
Related reagents
Click Chemistry
- Biotinylated-Alkyne (ES100)
- GDP-Azido-Fucose (ES101)
- CMP-Azido-Sialic Acid (ES102)
- UDP-Azido-GalNAc (ES103)
- UDP-Azido-GlcNAc (ES104)
Enzymes and Detection Reagents for CMP-C9-Biotin-Sialic Acid, ES201
- Various sialyltransferases
- Various neuraminidases/sialidases
- Streptavidin-HRP (DY998)
Data Examples
Titration of CMP-C9-Biotin-Sialic Acid on Asialofetuin
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Labeling and detection of N-glycans on fetal bovine fetuin. Asialofetuin (AF) was prepared from fetal bovine fetuin by treatment with Recombinant C. perfringens Neuraminidase, CF (Catalog # 5080-NM) and purified through size exclusion chromatography. Asialofetuin (8 µg) was incubated with indicated amounts of CMP-C9-Biotin-Sialic Acid (Catalog # ES201) with constant amount of Recombinant Human ST6GAL1 (aa 44-406) Protein (Catalog # 7620-GT) (0.1 µg) in 25 µl of 25 mM Tris, 10 mM MnCl2, pH7.5 buffer at 37oC for 30 minutes. The reactions were first separated on an SDS-PAGE and imaged by trichloroethylene (TCE) staining (upper panel). The separated proteins were then blotted to a nitrocellulose membrane and detected with conventional Streptavidin-HRP (Catalog # DY998) chemiluminescence reagents (lower panel). It is concluded that 0.1 nmol of CMP-C9-Biotin-Sialic Acid will achieve saturating labeling. |
Table 1. Enzyme selection for Glycan labeling
| Glycans to be labeled | Labeling enzymes (R&D Systems, Catalog# ) |
Neuraminidase (R&D Systems, Catalog# ) |
| N-Glycan | ST6Gal1 (7620-GT) | C. perfringens Neuraminidase (5080-NM) |
| ST3Gal4 | ||
| ST3Gal6 | ||
| O-Glycan | ST3Gal1 (6905-GT) | C. perfringens Neuraminidase (5080-NM) |
| ST3Gal2 (7275-GT) | ||
| ST6GalNAc1 (9154-GT) | ||
| ST6GalNAc2 (6468-GT) | ||
| ST6GalNAc3 | ||
| ST6GalNAc4 (6876-GT) | ||
| Polysialic acid (PSA) | ST8Sia1 (6716-GT) | M. viridifaciens Neuraminidase (5084-NM) |
| ST8Sia2 (6590-GT) | ||
| ST8Sia4 (7027-GT) | ||
| ST8Sia6 (9587-GT) |
Specifications
Product Datasheets
Assay Procedure
Sample Protocol for Labeling & Detecting Sialoglycans with Biotinylated Sialic Acid
Protocols are guidelines. Parameters need to be optimized by end users.
Materials
Assay Buffer: 25 mM Tris, 10 mM MnCl2, pH 7.5
Sample glycoprotein
Recombinant sialyltransferases
CMP-C9-Biotin-Sialic Acid (R&D Systems®, Catalog # ES201)
Streptavidin-HRP (R&D Systems®, Catalog # DY998)
SDS-PAGE and Western Blot reagents or equivalent
TBST buffer: 25 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.5
10% Fat Free Milk
Assay Procedure
- Prepare reaction mixture by combining a sample glycoprotein (ideally between 1 to 10 µg), 0.5 µg of a recombinant sialyltransferase, 0.1 µg of recombinant neuraminidase, 1 nmol CMP-C9-Biotin-Sialic Acid, in the Assay Buffer with the final volume of 25 μL.
- Prepare negative controls by combining a sample glycoprotein (ideally between 1 to 10 µg), 0.1 µg of recombinant neuraminidase, 1 nmol CMP-C9-Biotin-Sialic Acid in the Assay Buffer with the final volume of 25 μL.
- Incubate reactions and controls at 37 °C for 30 minutes.
- Separate the reactions and controls by SDS-PAGE.
- Blot the gel to a nitrocellulose membrane.
- Block the blot with 10% fat-free milk for 5 minutes.
- Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 30 minutes.
- Incubate the blot with 25 ng/mL Streptavidin-HRP in 30 mL TBST buffer for 30 minutes.
- Thoroughly wash the membrane with TBST buffer by changing buffer three times for a total of 45 minutes.
- Detect with commercial ECL (Enhanced Chemiluminescence) reagents.
Final Assay Conditions Per Reaction
CMP-C9-Biotin-Sialic Acid : 1 nmol
Recombinant sialyltransferase: 0.5 µg
Recombinant neuraminidase : 0.1 µg
Sample glycoproteins: 1-10 µg
FAQs
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