2-AB Labeling Kits-Sodium Cyanoborohydride
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 = 320 nm, em = 420 nm.
Number of samples Typically, a maximum of 15 individual analysis samples per set of labeling reagents.
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.
Labeling efficiency is typically > 85 % (depending on the sample). Label selectivity is essentially stoichiometric labeling.
Storage: Store at room temperature away from light. Keep away from heat, light sources and moisture. The reagents provided are stable for at least two years.
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
Each labeling reaction set consists of one vial of:
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Additional reagents and equipment required
Heating block, oven or similar dry heater (water bath cannot be used) set to 65°C
0 Centrifugal evaporator (e.g. 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 procedure (including optional post-labeling sample clean-up) typically takes 4-5 hours:
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Reductive amination reaction
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 Schiff base.
The Schiff base imine group is chemically reduced to produce stable labeled glycans.
Glycans are labeled with 2-aminobenzamic acid (2-AB) by reductive amination.
Label Agreement Outline
Glycan Labeling Kits are designed for fluorophore or chromophore labeling of free reducing ends of glycans. Labeled glycans can then be subjected to high-sensitivity fluorescent detection or monitored by UV absorbance during various chromatographic and structural sequence analyses. These include HPLC column chromatography and sequencing using exoglycosidases
The outline of the tagging procedure is as follows:
1 Prepare glycans: Prepare glycan samples by removing contaminants that may interfere with the labeling procedure, such as salts and detergents.
2 Drying the glycans: Place the sample in a reaction vial and air dry.
3 Prepare labeling reagents: Prepare fresh dye labeling solution by mixing the reagents in the kit.
4 Add labeling reagent to glycans: Add a small amount of labeling solution to each sample.
5 Incubation: Incubate the sample to allow the labeling reaction to proceed.
6 Post-labeling clean-up: After incubation, if needed (depending on subsequent analysis procedure), remove excess labeling reagent using a simple clean-up procedure.
7 Store or analyze labeled glycans: The labeled glycans are now ready for analysis.
Sample preparation
The glycan sample to be labeled, whether purified or a glycan mixture, must contain free reducing ends, be free of particles and salts, and be present in a volatile solvent system (preferably pure water).
The following substances may interfere with the labeling reaction and must first be removed from the glycan sample
Non-volatile solvents
Non-volatile salts, especially transition metal ions
Detergent
Dyes and stains such as Coomassie blue
The standard sample preparation protocol is as follows:
1 Purify glycans: If necessary, use one of these strategies to remove non-carbohydrate contaminants from the sample outlined in.
2 Transfer the sample to the reaction vial: For glycan pools obtained from typical glycoproteins, the sample volume should be between 100 Namol - 50 nmole. Using a single pure glycan allows labelling and detection down to 5 molar sodium in subsequent HPLC analysis. Suitable reaction vials include small polypropylene microcentrifuge tubes and test tubes for PCR work.
3 Drying the sample: Ideally, the sample should be dried using a centrifugal evaporator. If this is not possible, then freeze-drying (lyophilization) can be used sparingly (especially ensuring that the sample is dried to the quality of the bottom of a small, compact vial). Do not expose samples to high temperature (> 28°C) or extreme pH conditions, as these conditions can lead to acid-catalyzed loss of sialic acid (high temperature, low pH) or epimerization reduction endpoints of glycans (at high at the pH value).
4 Prepare the DMSO‑acetic acid mixture: Add 150 µL of glacial acetic acid to the DMSO vial and mix by pipette motion. Carefully tap or flick the contents of the top half of the ampoule, then open the ampoule, then carefully open the ampoule. If the DMSO is frozen, gently heat the vial (before opening) to between 30oC and 65oC in an oven or heating block.
5Add dye: Add 100 µL of DMSO-acetic acid mixture to a vial of 2-AB (2-aminobenzamic acid) for staining and mix until dye is dissolved.
6 Add the reducing agent: Add the dissolved dye to the vial of sodium cyanoborohydride (reducing agent), then mix the pipette action until the reducing agent is completely dissolved, making the final labeling reagent. If the reducing agent is difficult to dissolve, warm the vial gently in a 65oC incubator for up to 4 minutes or place on a heating block at this temperature and mix by pipette motion. If the undissolved reducing agent is still visible, add 10 µL of purified water to the vial and mix. Protect labeling reagents from moisture and use within 60 minutes.
7 Add labeling reagent to samples: Add 5 µL of labeling reagent to each dried glycan sample, cap the microtube, mix thoroughly, and tap gently to ensure the labeling solution is at the bottom of the vial.
8 Incubation: Place reaction vials in a heat block, sand tray, or dry oven set to 65°C and incubate for 3 hours. Incubation must be done in a dry environment. Use an oven or dry block - please do not use a water bath. The sample must be completely dissolved in the labeling solution for effective labeling. Complete lysis is encouraged Samples can be vortexed for 30 minutes after the 65°C incubation begins, then the incubation continues. In most cases, the incubation time can be shortened to 2 hours or extended to 4 hours without significantly altering the results of the labeling reaction.
9 Centrifuge and cool: Remove the samples after the incubation period, centrifuge the microtubes briefly, 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. An overview is below.
High Performance Liquid Chromatography: 2‑AB-labeled glycan mixtures can be separated and analyzed by a variety of HPLC (High Pressure Liquid Chromatography) methods
Chromatographic columns are particularly powerful tools for the purification and analysis of labeled oligosaccharides from complex glycan mixtures.
Enzymatic analysis
High-purity, sequencing-grade enzymes such as exoglycosidases, suitable for N- and O-linked labeled glycans are available from many 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.
References
1 Bigger, JC; Patel, TP; Bruce, JA; Goulding, PN; Charles, SM; Non-selective and efficient fluorescent labeling". Analytical Biochemistry 230: 229‑238
2 Trick, GR; Rudd, PM; Dr. Wing; Prime, SB; Dwek, RA (1996) A Rapid, High-Resolution, High-Performance Liquid Chromatographic Method for Separation of Glycan Mixtures and Analytical Oligosaccharide Profiling". Analytical Bio Chemistry 240: 210‑226
3 Townsend, RR; Lipneunas, PH; Bigger, C. Ventom, A. Parek (1996) Multimodal high performance liquid chromatography of fluorescently labeled oligosaccharides for glycoproteins". Analytical Biochemistry 239: 200‑207
4 Guidelines for High Resolution HPLC Carbohydrate Analysis (
5 Enzyme Selection Guide
6 Guidelines for Glycan Purification
7 Mr. Hardy (1997) Labeling of glycans with the fluorophores 2-aminobenzamide and anthranilic acid in "Techniques in Glycobiology", edited by Townsend, RR and Hotchkiss, AT. Marcel Dekker Inc, New York.
8 TechNote: Analysis of 2‑AB (2‑aminobenzamic acid)-labeled glycans