Glycoprofile™ Labeling Kits

The GlycoProfile™ Labeling Kits are designed for efficient labeling of N-linked, O-linked and glycosylphosphatidylinositol (GPI) anchored glycans using your choice of 2-aminobenzamide (2-AB) or 2-aminobenzoic acid (anthranilic acid; 2-AA) ^1,2. These small fluorophores increase the spectral absorption of glycans, improving detection in HPAE (high-performance anion exchange chromatography),^3-6 HPLC^4-7, and ESI-MS (electrospray mass spectroscopy) methods. Binding is robust and the resulting dye-glycan conjugates are stable, with no degradation during post-labeling analysis. 2-AB is recommended for downstream analysis by HPAE, HPLC and ESI-MS, although it is slightly less sensitive than 2-AA. 2-AA is recommended for SDS-PAGE, and is also suitable for HPAE, HPLC and ESI-MS.

Figure 1. Labeling of carbohydrates with 2-AB

Features and Benefits

• Each kit is complete, containing 2-AA or 2-AB dye, reductant and solvents.
• Sufficient for the preparation of up to 36 samples of mixed glycans containing from 100 picomoles to 50 nanomoles of purified glycans. With a single pure glycan, as little as 5 picomoles may be labeled and detected in subsequent HPLC analysis.

Note: Labeling of glycans with 2-AB is covered under US Patent No 5,747,347 and its foreign equivalents.

GlycoProfile™ Glycan Clean-Up Cartridges are recommended for use with the GlycoProfile Labeling kits for post-labeling cleanup. Glycans adsorb to the membrane while excess dye, mono- and disaccharides and other hydrophobic contaminants pass through. Purified labeled glycans are eluted with water and are ready for subsequent enzymatic digestion or analysis.

References

1. Bigge J, Patel T, Bruce J, Goulding P, Charles S, Parekh R. 1995. Nonselective and Efficient Fluorescent Labeling of Glycans Using 2-Amino Benzamide and Anthranilic Acid. Analytical Biochemistry. 230(2):229-238. https://doi.org/10.1006/abio.1995.1468

2. Hardy M. Glycan labeling with the fluorophores 2-aminobenzamide and anthranilic acid, Techniques in Glycobiology, Dekker, Inc. NY (1997)..

3. Delaney J, Vouros P. 2001. Liquid chromatography ion trap mass spectrometric analysis of oligosaccharides using permethylated derivatives. Rapid Commun. Mass Spectrom.. 15(5):325-334. https://doi.org/10.1002/rcm.230

4. Anumula KR, Dhume ST. 1998. High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiology. 8(7):685-694. https://doi.org/10.1093/glycob/8.7.685

5. Townsend R, Lipniunas PH, Bigge C, Ventom A, Parekh R. 1996. Multimode High-Performance Liquid Chromatography of Fluorescently Labeled Oligosaccharides from Glycoproteins. Analytical Biochemistry. 239(2):200-207. https://doi.org/10.1006/abio.1996.0315

6. Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM. 2002. An Analytical and Structural Database Provides a Strategy for Sequencing O-Glycans from Microgram Quantities of Glycoproteins. Analytical Biochemistry. 304(1):70-90. https://doi.org/10.1006/abio.2002.5619

7. Guile GR, Rudd PM, Wing DR, Prime SB, Dwek RA. 1996. A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles. Analytical Biochemistry. 240(2):210-226. https://doi.org/10.1006/abio.1996.0351

8. Rudd PM, Mattu TS, Zitzmann N, Mehta A, Colominas C, Hart E, Opdenakker G, Dwek RA. 1999. Glycoproteins: Rapid Sequencing Technology for N-linked and GPI Anchor Glycans. Biotechnology and Genetic Engineering Reviews. 16(1):1-22. https://doi.org/10.1080/02648725.1999.10647969