2-AB Labeling Kits-2-Picoline Borane
Product description
Application: For labeling free glycans with 2-aminobenzamic acid (2-AB).
Description: This kit contains reagents for coupling dyes to the free reducing end of glycans by reductive amination.
Dye Properties: Mass = 136.15
Fluorescence, ex = 250 nm, em = 428 nm.
Number of samples 12 individual assay samples per set of labeling reagents (24 samples total for kit)
Sample amounts per sample ranged from 25 pmol to 25 nmol glycans.
Appropriate samples can label any purified glycan with free reducing ends.
No detectable (< 2 mol %) loss of structural integrity sialic acid, fucose, sulfate or phosphate.
Storage: Store at room temperature away from light. Keep away from heat, light sources and moisture. The reagents are stable for at least two years from the date of manufacture.
Shipping: This product can be shipped at ambient temperature.
Handling: Make sure any glass, plastic or solvent used is free of glycosidases and environmental carbohydrates. Use powder-free gloves for all sample handling procedures and avoid contaminating environmental carbohydrates. All steps involving labeling reagents must be performed on glassware and plasticware in a dry environment. Once opened, the contents of a single vial should be used immediately and discarded in accordance with local safety regulations after overdose.
Safety: For Research Use Only. Not for human or medicinal use
Kit contents
|
Additional reagents and equipment required
Milli Q water or similar
Heat block, oven or similar dry heater (no water bath) set to 65°C
Centrifugal evaporators (eg Savant, Heto or similar)
Reaction vials (eg polypropylene microcentrifuge vials)
NOTE: Additional reagents are required if optional post-label sample cleanup is performed
Timeline of labels
The labeling process takes 2 hours, while the actual labeling incubation takes only 1 hour.
|
Labeling method
1 Purify glycans: If necessary, remove non-carbohydrate contaminants from the sample.
2 Transfer the sample to a reaction vial: The kit is designed to label up to 25 nmol of glycans per reaction. In subsequent HPLC analysis, each reaction can label and detect as low as 5 pmols of a single pure glycan. Suitable reaction vials include small polypropylene microcentrifuge tubes and PCR work tubes.
3Dry the sample and redissolve in 10 µL of water: If the sample volume exceeds 10 µL, dry the sample. If the sample needs to be dried, it should be dried using a centrifugal evaporator. If this is not possible, freeze-drying (lyophilization) can be used with caution (especially ensuring that the sample dries to a small, dense mass at the bottom of the vial). Do not subject samples to high temperatures (>28°C) or extreme pH, as these conditions can lead to acid-catalyzed loss of sialic acid (high temperature, low pH) or epimerization of the reducing end of the glycan (in high under pH conditions). After the sample has dried, redissolve the glycans in 10 μL of water.
4 Prepare the labeling reagents: Add 150 µL of kit components 30% acetic acid in DMSO to a vial of dye (and mix by pipetting until the dye dissolves. Sometimes heat (30‑60°C) is required to help dissolve the dye. Add 150 µL of dissolved dye solution is transferred to a bottle of reducing agent and mixed by pipette motion until reducing agent is dissolved. Heat (30‑60°C) is sometimes required to help dissolve reducing agent.
5Add labeling reagent to samples: Add 10 µL of labeling reagent to each glycan sample, cap the microtube, mix well, and tap gently to ensure the labeling solution is at the bottom of the vial.
6 Incubation: Place reaction vials in a heating block, sand tray, or dry oven set to 65°C and incubate for 1 hour. The sample must be completely dissolved in the labeling solution for effective labeling. Complete lysis is encouraged Samples can be vortexed for 10 minutes after incubation at 65°C begins, then incubation continues.
7 Centrifuge and cool: After the incubation period, remove the samples, briefly centrifuge the microtubes, and allow them to cool completely to room temperature.
Analysis of 2AB-labeled glycans
2‑AB-labeled glycans can be studied by a number of different analytical methods, including HPLC, gel electrophoresis, and mass spectrometry.
high performance liquid chromatography
2‑AB-labeled glycan mixtures can be analyzed by various HPLC (High Pressure Liquid Chromatography) methods:
Analysis type
Separation of charged and neutral glycans
Profiling of neutral and charged glycans Separation of neutral glycans
N columns are particularly powerful tools for the purification and analysis of labeled oligosaccharides from complex glycan mixtures.
Enzymatic analysis
High-purity sequencing-grade enzymes (eg, exoglycosidases) suitable for structural analysis of N- and O-linked labeled glycans are available from a number of companies.
Be especially careful when choosing a glycosidase, choosing those formulas that match your specific application. For example, some enzymes and enzyme buffers contain interference by certain types of assays.
Mass Spectrometry and Electrophoresis
Labeled glycans can also be analyzed by mass spectrometry, electrophoresis, and various types of spectroscopy.
Reductive amination reaction
The labeling reaction involves a two-step process
1. Schiff base formation.
This requires glycans with free reducing ends that are in equilibrium between closed (cyclic) and open (acyclic) forms. The primary amino group of the dye nucleophilically attacks the carbonyl carbon of the acyclic reducing terminal residue to form a partially stabilized Schiff base.
2. Reduce the Schiff base.
The Schiff base imine group is chemically reduced to produce stable labeled glycans.
Labeling of glycans with 2-aminobenzamic acid (2-AB) by reductive amination