Terminal GlcNAc can be detected on O- or N-Linked glycans. Glycans are removed by enzyme treatment. Specific transferases can be used to incorporate Azido-GlcNAc at suitable open positions. Azido-Sialic Acid can then be detected using Biotinylated Alkyne in a click chemistry reaction.
Sample Data
Assessing the Presence of open GlcNAc Incorporation Sites Using GlcNAc Transferases. Total protein labeling is shown in the upper panel. Streptavidin-HRP detection is shown on the lower panel. UDP-Azido-GlcNAc is only incorporated into D-Fetuin (rcpNeuraminidase–treated Fetuin). The results indicate untreated Fetuin does not contain open sites for GlcNAc, whereas D-Fetuin contains incorporation sites that differ depending on enzyme specificity. In addition MW = molecular weight markers.
In each reaction, 10 µg Fetuin or D-fetuin sample, 0.3 nmol of UDP-Azido-GlcNAc, and 2 µg of the indicated GlcNAc transferases were mixed in 50 µL of 25 mM Tris supplemented with 10 mM of MnCl2 and 150 mM NaCl at pH 7.5. The reaction was incubated at 37 °C for a minimum of 20 minutes. The reactions were then conjugated with 0.1 mM Biotinylated Alkyne in the presence of 2 mM of Ascorbic Acid and 0.1 mM of CuCl2. The reactions were incubated at room temperature for 30 minutes. The reactions were then separated with 12% SDS-PAGE and blotted to a nitrocellulose paper and detected with Streptavidin-HRP.
Fetuin, from Sigma Aldrich, was further purified with Gel filtration column. Fetuin was treated with rcpNeuraminidase to prepare D-fetuin
Product Specifications for UDP-Azido-GlcNAc
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 GlcNAc Detection on Core-1 O-glycan
Protocols are guidelines. Parameters need to be optimized by end users.
Materials
Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl2, pH 7.5.
1. Prepare a reaction mixture by combining 5 µg of Protein Sample, 1 µg of rhGCNT1 in the presence of 1 nmol of UDP-Azido-GlcNAc in the Assay Buffer with the final volume of 25 µL.
2. Prepare negative controls according to step 1 but omit Protein Sample or rhGCNT1.
3. Incubate all the reactions and the 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 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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