2-AA Labeling Kit-2-Picoline Borane
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 (glycan-dye conjugate) = 250 nm, em = 425 nm.
For maximum sensitivity of detection we recommend an excitation wavelength of 250 nm.
Number of Samples：12 separate analytical samples per set of labeling reagents (24 samples in total per kit)
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 Selectivity：Essentially stoichiometric labeling.
Storage：Store at room temperature in the dark. Protect from sources of heat, light, and moisture.
Shipping: The product can be shipped at ambient temperature.
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 kit contains two labelling reaction sets. Each labeling reaction set consists of one vial of each of the following:
Item | Quantity |
2-AA Dye (2-aminobenzoic acid) | 7.5 mg |
2PB reductant (2-picoline borane) | 16.5 mg |
30% acetic acid in DMSO | 500 µl |
Additional Reagents and Equipment Required
Milli Q water or similar
Heating block, oven, or similar dry heater (a water bath cannot be used) set at 65°C
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 (see Section on Sample Cleanup)
Time Line for Labeling
labeling procedure takes 2 hours with just 1 hour for the actual labelling incubation.
Procedure | Time | Elapsed Time (hours) |
Transfer samples to reaction tube and dry | 30 min | 0.5 |
Add water to samples | 15 min | 0.75 |
Make up and add labeling reagent | 15 min | 1 |
Incubate samples with reagent | 1 hour | 2 |
Labeling Method
1，Purify the glycans
If necessary, remove non-carbohydrate contaminants from the samples
2，Transfer sample to reaction vial
The kit is designed to label up to 25 nmols of glycans per reaction. With a single pure glycan as little as 5 picomoles per reaction 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 and resuspend in 10 µL of water
Dry down the samples if the volume of the sample exceeds 10 µL.
If the samples need to be dried down then this should be done 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°C) 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).Once the samples are dry then redissolve the glycans in 10 µL of water.
4，Prepare the labelling reagent
Add 150 µl of kit componentto a vial of dyeand mix by pipette action until the dye is dissolved. Sometimes heat (30-60°C) is required to help
dissolve the dye.
Transfer the 150 µL of dissolved dye solution to a vial of reductant (LT-PB-01) and mix by pipette action until the reductant is dissolved. Sometimes heat (30-60°C) is required to help dissolve the reductant.
5，Add labeling reagent to samples
Add 10 µl of labeling reagent to each glycan sample, cap the microtube, mix thoroughly, and then gently
tap to ensure the labeling solution is at the bottom of the vial.
6，Incubate
Place the reaction vials in a heating block, sand tray, or dry oven set at 65°C and incubate for 1 hour.
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°C incubation then
the incubation continued.
7，Centrifuge and cool
After the incubation period remove the samples, centrifuge the microtubes briefly, and then allow them
to cool completely to room temperature.
Analysis of 2-AA-Labeled Glycans
2-AA labeled glycans may be studied by a number of different analytical methods including HPLC, gel electrophoresis, and mass spectrometry.
HPLC Analysis
labeled glycan mixtures may be separated and analysed by a variety of HPLC (high pressure liquid chromatography)
Enzymatic Analysis
High purity, sequencing grade enzymes (e.g. exoglycosidases) suitable for structural analysis of both N- and
O-linked LudgerTag™ labeled glycans are available from a number of companies.
When selecting glycosidases be especially careful to choose those with formulations that are compatible with
your particular application. For example, some enzymes and enzyme buffers have components that interfere
with certain types of analysis. Please call us for guidance in selecting enzymes and reaction conditions for
your work.
Mass Spectrometry and Electrophoresis
labeled glycans may also be analysed by mass spectrometry, electrophoresis, and various types of spectroscopy. Please call us for advice on the analysis conditions most suitable for your intended analyses.
The Reductive Amination Reaction
The labeling reaction involves a two step process (see Figure 1):
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-aminobenzamide acid (2-AA) by reductive amination.