CMP-Azido-Sialic Acid

Activated Sugar
Catalog # Availability Size / Price Qty
ES102-100
Product Details
Procedure
Citations (1)
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CMP-Azido-Sialic Acid Summary

 

CMP-Azido-Sialic Acid Structure

Formula

C20H30N7O15P

Molecular Weight

639.46 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 sialic acid without expensive specialized equipment!

Applications

  • For in vitro enzymatic incorporation of azido-sugars into specific, targeted glycans.
  • Detecting the presence or absence of sialated glycans.
  • Labeling terminal sialoglycans on proteins and lipids.

Key Features and Benefits

  • Can detect terminal sialoglycans without specialized, expensive equipment.
  • Can be introduced to proteins and lipids via various sialyltransferases.
  • Can be conjugated to desired reporter molecules, such as biotin, via click chemistry.
  • Can be detected via Western blot, ELISA, and flow cytometry, depending on the type of reporter molecule.
  • Has minimal side effects 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 CMP-Sialic-Acid, ES102

Sialyltransferases

Sialation Link Specificity

ST3 beta-Gal alpha-2,3-Sialyltransferase 1/ST3GAL1 Adds Sialic Acid to Galactose
ST3 beta-Gal alpha-2,3-Sialyltransferase 2/ST3GAL2 Adds Sialic Acid to Galactose
ST3GAL5/GM3 Synthase Adds Sialic Acid to Galactose
ST6 Gal Sialyltransferase 1/ST6GAL1 Adds Sialic Acid to Galactose
ST6 Sialyltransferase 2/ST6GALNAC2 Adds Sialic Acid to GalNAc
ST6GALNAC4 Adds Sialic Acid to GalNAc
ST6 GalNAc alpha-2,6-sialyltransferaseV/ST6GALNAC5 Adds Sialic Acid to GalNAc
ST6 Sialyltransferase 6/ST6GALNAC6 Adds Sialic Acid to GalNAc
ST6 Gal Sialyltransferase 2/ST6GAL2 Adds Sialic Acid to Galactose
ST6 Sialyltransferase 1/ST6GALNAC1 Adds Sialic Acid to GalNAc
ST8 alpha-2,8-Sialyltransferase 8A/ST8SIA1 Adds Sialic Acid to Sialic Acid
ST8 alpha-2,8-Sialyltransferase 8B/ST8SIA2 Adds Sialic Acid to Sialic Acid
ST8 alpha-2,8-Sialyltransferase 4/ST8SIA4 Adds Sialic Acid to Sialic Acid

 

 

 

Schematic

Terminal sialic acid
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Terminal sialic acid can be detected on O- or N-linked glycans. Sialic acid is removed using a Sialidase. The specificity of Sialyltransferases allows you to add Azido-Sialic Acid to open positions. Azido-Sialic Acid can be detected using Biotinylated Alkyne in a click chemistry reaction.

Data Examples

Labeling and detection of O-glycans on Bovine Fetuin
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Streptavidin-HRP detection of rhST3GAL1-mediated CMP-Azido-Sialic Acid  incorporation
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Labeling and detection of sialic acid on O-glycans on Bovine Fetuin. Fetuin (left side of the molecular marker; MW) and Desialylated Fetuin (right side of the molecular marker) were incubated with increasing amount of rhST3GAL1 (5, 25, 50, 250 ng) and 0.5 nmol CMP-Azido-Sialic Acid. Fetuin was treated with rcpNeuraminidase to prepare desialylated Fetuin. The reactions were then conjugated with Biotinylated Alkyne and separated by SDS-PAGE. The separated proteins were then blotted to a nitrocellulose membrane and detected with conventional Streptavidin-HRP chemiluminescence reagents. A. Total protein staining. B. Streptavidin-HRP detection of rhST3GAL1-mediated CMP-Azido-Sialic Acid incorporation. No incorporation of CMP-Azido-Sialic Acid is detected in fully sialted Fetuin. In contrast, strong labeling indicates CMP-Azido-Sialic Acid is incorporated into desialated Fetuin.

Data adapted from Wu et al., (2015) Carbohydrate Res. 412:1-6.

Product Datasheets

Preparation and Storage

Shipping
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

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 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

Citation for CMP-Azido-Sialic Acid

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

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