2-AA Labeling Kit-Sodium Cyanoborohydride
Product Description
Application：For labeling of free glycans with 2-aminobenzoic acid (2-AA).
Description：The kit contains reagents for the conjugation of dye to the free reducing end of the glycan by a reductive amination reaction.
Dye Properties：Mass = 137，Fluorescence, ex (free dye) = 320 nm, ex (glycan-dye conjugate) = 360 nm，em = 420 nm.
For maximum sensitivity of detection we recommend an excitation wavelength of 360
nm
Amount of Sample：From 25 pmol up to 25 nmol glycans per sample.
Suitable Samples：Any purified glycans with free reducing termini can be labeled.
Structural Integrity：No detectable (< 2 mole per cent) loss of sialic acid, fucose, sulfate, or phosphate.
Labeling efficiency：Typically > 85 % (dependent on sample).
Labeling Selectivity：Essentially stoichiometric labeling.
Storage: Store at room temperature in the dark. Protect from sources of heat, light, and moisture. T
Handling: Ensure that any glass, plasticware or solvents used are free of glycosidases and environmental carbohydrates. Use powder-free gloves for all sample handling procedures and avoid contamination with environmental carbohydrate. All steps involving labeling reagents must be performed in a dry environment with dry glassware and plasticware. Once individual vials of reagents are opened, their contents should be used immediately and excess then discarded according to local safety rules.
Safety: For research use only. Not for human or drug use.
Kit Contents
Each labeling reaction set consists of one vial of:
Item | Quantity |
2-AA Dye (2-Aminobenzoic acid) | 5 mg |
DMSO | 350 μL |
Acetic acid | 200 μL |
Sodium cyanoborohydride (Reductant) | 6 mg |
Additional reagents and equipment required
Heating block, oven, or similar dry heater (a water bath cannot be used) set at 65 ℃
Centrifugal evaporator (e.g. Savant, Heto, or similar)
Reaction vials (e.g. polypropylene microcentrifuge vials)
Note: Further reagents are required if doing the optional post-labeling sample cleanup
Timeline of labels
The labeling procedure (including optional post-labeling sample clean-up) typically takes 4-5 hours:
Procedure | Time | Elapsed Time (hours) |
Transfer samples to reaction tube and dry | 30 min | 0.5 |
Make up and add labeling reagent | 15 min | 0.75 |
Incubate samples with reagent | 3 hours | 3.75 |
Post-labeling cleanup | 1 hour | 4.75 |
Reductive amination reaction
1.Schiff's base formation.
This requires a glycan with a free reducing terminus which is equilibrium between the ring closed (cyclic) and ring open (acyclic) forms. The primary amino group of the dye performs a nucleophilic attack on the carbonyl carbon of the acyclic reducing terminal residue to form a partially stable Schiff's base.
2.Reduction of the Schiff's base.
The Schiff's base imine group is chemically reduced to give a stable labeled glycan.
Figure 1: Labeling of a glycan with 2-aminobenzoic acid (2-AA) by reductive amination.
Outline of Labeling Protocol
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 labeling procedure is as follows:
1.Prepare the glycans.Prepare the glycan samples by removing contaminants such as salts and detergents that could interfere with the labeling procedure.
2.Dry the glycans
Place the samples in reaction vials and dry down.
3.Prepare labeling reagent
Prepare fresh dye labeling solution by mixing reagents in the kit.
4.Add labeling reagent to glycans
Add a small amount of labeling solution to each sample.
5.Incubate
Incubate the samples to allow the labeling reaction to progress.
6.Post-labeling cleanup
After incubation, if required (depending on the subsequent analysis procedures), remove the excess labeling reagents using a straightforward cleanup procedure.
7.Store or analyse the labeled glycans
The labeled glycans are now ready for analysis.
Sample Preparation
The glycan sample to be labeled, whether a purified glycan or a glycan mixture, must contain a free reducing terminus, be particle and salt-free, and be presented 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, in particular transition metal ions
Detergents
Dyes and stains such as Coomassie Blue
The standard sample preparation protocol is as follows:
1.Purify the glycans
If necessary, use one of these strategies to remove non-carbohydrate contaminants from the sample outlined in.
2.Transfer sample to reaction vial
The amount of sample should be in the range 100 picomoles - 50 nanomoles for a glycan pool obtained from a typical glycoprotein. With a single pure glycan as little as 5 picomoles can be labeled and detected in subsequent HPLC analysis. Suitable reaction vials include small polypropylene microcentrifuge tubes and tubes for PCR work.
3.Dry the samples
Ideally, samples should be dried using a centrifugal evaporator. If this is not possible then freeze drying
(lyophilization) can be used with caution (in particular, ensure that the sample dries to a small, compact
mass at the very bottom of the vial).
Do not subject samples to high temperatures (> 28 oC) or extremes of pH as these conditions will result
in acid catalysed loss of sialic acids (high temperatures, low pH) or epimerization of the glycan reducing
terminus (at high pH).
Preparation of Labeling Reagent
Prepare fresh labeling reagent as follows:
1.Prepare a DMSO-acetic acid mixture
Add 150 μL glacial Acetic Acid to the vial of DMSO and mix by pipette action. Open the ampoules by carefully tapping or flicking to dislodge any contents in the upper half, then carefully break open the ampoule. If the DMSO is frozen then gently warm up the vial (before opening) in an oven or heating block to between 30℃and 65℃.
2.Add the dye
Add 100 μL of the DMSO-acetic acid mixture to a vial of 2-AA (2-Aminobenzoic Acid) Dye and mix until the dye is dissolved.
3.Add the reductant
Add the dissolved dye to a vial of Sodium Cyanoborohydride (reductant) and mix by pipette action until the reductant is completely dissolved to make the final labeling reagent.If the reductant is difficult to dissolve then gently warm the vial for up to four minutes in the 65℃ incubation oven or stand on a heating block at this temperature then mix by pipette action. If undissolved reductant is still visible add 10 μL pure water to the vial and mix.Protect the labeling reagent from exposure to moisture and use within 60 minute.
Labeling Reaction
1.Add labeling reagent to samples
Add 5 μL of labeling reagent to each dried glycan sample, cap the microtube, mix thoroughly, and then gently tap to ensure the labeling solution is at the bottom of the vial.
2.Incubate
Place the reaction vials in a heating block, sand tray, or dry oven set at 65℃ and incubate for 3 hours. The incubation must be performed in a dry environment. Use an oven or dry block - please do not use a water bath.The samples must be completely dissolved in the labeling solution for efficient labeling. To encourage complete dissolution the samples can be vortexed 30 minutes after the start of the 65℃ incubation then the incubation continued.In most cases, the incubation time can be shortened to 2 hours or extended up to 4 hours without
significantly changing the outcome of the labeling reaction.
3.Centrifuge and cool
After the incubation period remove the samples, centrifuge the microtubes briefly, then allow them to cool completely to room temperature.
Analysis of2AA-Labeled Glycans
2-AA labeled glycans may be studied by a number of different analytical methods including HPLC, gel electrophoresis, and mass spectrometry.2-AA labeled glycan mixtures may be separated and analysed by a variety of HPLC (high pressure liquid chromatography) methods.
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 Bigge, J.C.; Patel, T.P; Bruce, J.A.; Goulding, P.N.; Charles, S.M; Parekh, R.B. (1995) 'Non-selective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid'. Analytical Biochemistry 230: 229-238
2 Guile, G.R.; Rudd, P.M.; Wing, D.R.; Prime, S.B.; Dwek, R.A. (1996) 'A rapid and high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles'. Analytical Biochemistry 240: 210-226
3 Townsend, R.R.; Lipniunas, P.H.; Bigge, C.; Ventom, A.; Parekh, R. (1996) 'Multimode high-performance liquid chromatography of fluorescently labeled oligosaccharides from glycoproteins'. Analytical Biochemistry 239: 200-207
4 LudgerSep High Resolution HPLC Carbohydrate Profiling Guide (Cat # LS-GUIDE-01)
5 Ludger Enzyme Selection Guide (Cat # EZ-GUIDE-01)
6 LudgerClean Glycan Cleanup Guide (Cat # LC-GUIDE-01)
7 Hardy, M.R. (1997) ‘Glycan labeling with the fluorophores 2-aminobenzamide and anthranilic acid’ in ‘Techniques in Glycobiology’, edited by Townsend, R.R and Hotchkiss, A.T.. Marcel Dekker Inc, New York.
8 Ludger Technical Note # TN-AA-01: Analysis of 2-AA (2-aminobenzoic acid) labeled glycans
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