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 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.
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.
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