N-glycans are the most common type of glycosylation found on glycoproteins and play critical roles in regulating protein folding, stability, cell communication, and immune recognition. Subtle changes in N-glycan structures are often closely associated with various disease states, including cancer, infections, autoimmune disorders, and neurodegenerative conditions. Therefore, highly sensitive and accurate detection of free N-glycans is a key step in both glycobiology research and clinical translational studies.
However, N-glycans lack strong intrinsic signals and cannot be directly detected by fluorescence or efficiently ionized in mass spectrometry. To improve detection sensitivity and separation efficiency, chemical derivatization is commonly employed to label glycans prior to analysis. 2-AB (2-aminobenzamide) labeling is one of the most widely used derivatization techniques for free N-glycans. It significantly enhances the fluorescence signal of glycans, enabling high-sensitivity detection and quantification on UPLC and HPLC platforms, while also being compatible with certain mass spectrometry applications.
Product Overview
The 2-AB N-glycan kit from MtoZ Biolabs is a fluorescence labeling product specifically designed for glycan derivatization. By offering ready-to-use reagents, an optimized reaction system, and a standardized workflow, the kit significantly reduces operational complexity, enhances labeling efficiency, and improves data consistency, providing a more efficient and reliable tool for N-glycan detection experiments. Whether you are focused on biopharmaceutical quality control, disease mechanism studies, or fundamental glycan structure analysis, this product can bring substantial convenience and benefits to your research.
Product Details
|
Product Details |
Size |
Storage Conditions |
|
PNGase F |
100 µL/vial |
2-8℃ |
|
Digestion Buffer (7.5×) |
200 µL/vial |
2-8℃ |
|
Enhanced Digestion Buffer (7.5×) |
200 µL/vial |
2-8℃ |
|
2-AB Solution |
500 µL/vial |
-20℃ |
|
Reducing Agent Solution |
500 µL/vial |
-20℃ |
|
Purification Magnetic Beads |
10 mL/bottle |
2-8℃ |
|
Elution Buffer |
2 mL/vial |
2-8℃ |
|
Wash Buffer |
40 mL/bottle |
2-8℃ |
Protocol
The entire labeling workflow has been carefully optimized by the MtoZ Biolabs team to suit standard laboratory operating procedures. The main steps are as follows:
1. Sample Digestion
The 2-AB N-glycan kit includes two enzymatic digestion options: non-denaturing digestion and denaturing digestion. The key difference lies in the fact that some N-glycan sites are difficult to release under non-denaturing conditions, requiring the use of enhanced digestion buffer for effective cleavage.
(1) Non-Denaturing Digestion
Dilute the sample with water to 5 mg/mL. Take 24 µL of the diluted sample, add 4 µL of digestion buffer, then add 2 µL of PNGase F. Mix well and incubate at 50 °C for 5 minutes for enzymatic digestion. Then heat at 98 °C for 5 minutes for denaturation. Centrifuge at ≥14,000 rpm for 3-5 minutes, and transfer 25 µL of the supernatant to a new EP tube.
(2) Denaturing Digestion
Dilute the sample with water to 5 mg/mL. Take 24 µL of the diluted sample, add 4 µL of enhanced digestion buffer, mix well, and heat at 98 °C for 3 minutes to denature. Then add 2 µL of PNGase F, mix well, and incubate at 50 °C for 5 minutes for digestion. Heat again at 98 °C for 5 minutes. After returning to room temperature, centrifuge the sample to the bottom of the tube.
2. 2-AB Labeling
While centrifuging, prepare the labeling solution simultaneously. Mix the 2-AB solution, reducing agent solution, and formic acid solution at a 2:2:1 volume ratio to prepare the labeling solution. After thorough mixing, take 25 µL of the labeling solution and add it to the previously collected 25 µL supernatant or 30 µL enzymatic digestion mixture. Mix well and incubate at 50 °C for 15 minutes. For different sample quantities, adjust the labeling solution volume proportionally as needed.
3. Magnetic Bead Purification
(1) Binding
Take out the magnetic beads used for purification, vortex to mix thoroughly, and pipette 200 µL of the bead suspension into an EP tube. Place the tube on a magnetic rack, and after the solution has cleared, carefully discard the supernatant.
Remove the EP tube from the magnetic rack, add 50 or 55 µL of the labeled mixture into the tube containing the beads, and pipette up and down to mix.
Add 330 µL of acetonitrile and pipette 3-4 times to mix thoroughly.
(2) Washing
Place the EP tube on the magnetic rack. After approximately 30 seconds, once the solution has cleared, carefully discard the supernatant. Remove the EP tube from the magnetic rack and add 400 µL of wash buffer. Pipette to mix thoroughly. Return the EP tube to the magnetic rack. After approximately 30 seconds, when the solution has cleared, discard the supernatant.
Repeat the above washing step once more.
(3) Elution
Remove the EP tube from the magnetic rack and add 40 µL of elution buffer. Pipette to mix thoroughly and incubate at room temperature for 2-3 minutes. Place the tube back on the magnetic rack. After about 30 seconds, when the solution has cleared, carefully transfer 40 µL of the supernatant to a new EP tube. Add 60 µL of acetonitrile and mix well. Centrifuge at ≥14,000 rpm for 3 minutes, then transfer the supernatant to an injection vial.
Note: It is recommended to dispense liquids along the sidewall of the tube above the magnetic beads to help flush the beads smoothly to the bottom of the tube.
Features and Benefits
1. Ready-to-Use Formulation
Key components are pre-configured, eliminating the need for manual preparation. This simplifies the workflow and reduces operational error.
2. Broad Platform Compatibility
Labeled products are suitable for mainstream chromatographic platforms such as UPLC and HPLC, and are also compatible with glycan databases and spectral library-based analysis.
3. Mild Reaction Conditions with High Efficiency
The labeling reaction is rapidly completed at moderate temperatures, making it suitable for various glycan structures with minimal degradation or side reactions.
4. Suitable for Diverse Sample Types
Applicable to glycans extracted from cells, serum, and tissue samples, meeting the diverse needs of basic research and drug development.
5. High Experimental Reproducibility
Validated by internal quality control, the kit provides consistent labeling efficiency and detection signals, supporting reliable multi-batch sample comparison.
Applications
The 2-AB N-glycan kit is broadly applicable to a variety of fields, including biopharmaceutical development, basic research, and bioanalytical studies, such as:
1. Characterization of Glycosylation in Antibody-Based Drugs
Labeling and analyzing N-glycan structures in monoclonal antibodies or fusion proteins to support consistency evaluation and quality control of biopharmaceuticals.
2. Disease Biomarker Discovery
Comparing glycan expression patterns in serum, cell, or tissue samples under different disease states to identify potential biomarkers.
3. Glycoengineering Research
Assessing glycan structures produced by genetically engineered cells for the optimization of glycosylation pathways.
4. Fundamental Glycomics and Systems Biology
Exploring glycan distributions and their biological functions across species, tissues, and developmental stages.
FAQs
Q1: Does This Kit Include Glycan-Releasing Enzymes?
A1: No, it does not. Users should release N-glycans using enzymes such as PNGase F prior to use. This kit is specifically designed for labeling free N-glycans.
Q2: Are Any Special Instruments Required during the Reaction?
A2: No special instruments are needed. Standard laboratory heating blocks or water baths are sufficient to meet the reaction conditions.
Q3: Does the Kit Support High-Throughput Sample Processing?
A3: Yes, it does. The kit is designed for parallel processing of multiple samples and is compatible with 96-well plate operations.
Q4: Can the Labeled Glycans Be Used for Mass Spectrometry Analysis?
A4: Yes. Glycans labeled with 2-AB can achieve good ionization efficiency on certain MS platforms, although the kit is primarily optimized for chromatographic-fluorescence detection methods.
Q5: Are the Labeled Samples Stable?
A5: Yes. The labeled products can be stored at -20°C, avoiding repeated freeze-thaw cycles, and remain stable for several weeks.
