Glycans are removed by enzyme treatment. Specific transferases can be used to incorporate Azido-GalNAc at suitable open positions. The incorporation Azido-GalNAc can then be detected using Biotinylated Alkyne in a click chemistry reaction.
Sample Data
Labeling Asialofetuin with GALNT2. Lane 1 and 2 shows UDP-Azido-GalNAc transferred to asialofetuin by rhGALNT2. Lane 3 was a negative control in the absence of UDP-Azido-GalNAc.
In each reaction, 5 µg of Asialofetuin (Sigma Aldrich), 0.5 nmol of UDP-Azido-GlcNAc, 1 µg rhGALNT2 and 1 µg of rE. faecalis O-Glycosidase was mixed in 50 µL of 25 mM HEPES supplemented with 10 mM of MnCl2 and 150 mM NaCl at pH 7.5. The reactions were incubated at 37°C for 60 minutes. The reactions were then conjugated, at room temperature for 30 minutes, with 1.0 nmol of Biotinylated Alkyne in the presence of 100 nmol of Ascorbic Acid and 5 nmol of CuCl2 for a final volume of 60 µL. The reactions were then separated with 12% SDS-PAGE and blotted to a nitrocellulose paper and detected with Streptavidin-HRP.
Product Specifications for UDP-Azido-GalNAc
Species
Multi-Species
Preparation & Storage
Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
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 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.
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.
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