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N-Glycan Sequencing Kit
The N-Glycan Sequencing Kit consists of seven well characterized, highly pure, recombinant exoglycosidase enzymes selected to simplify the process of characterizing typical N-linked glycan structures.
Recombinant enzymes with no detectable endoglycosidase or other exoglycosidase contaminating activities
Compatible with N-linked glycans released from a variety of CHO and murine derived antibodies, as well as N-linked glycans released from other glycoproteins
Simultaneous digestion with other exoglycosidases
Optimal activity and stability for up to 12 months
Storage Temperature: 4°C
The N-Glycan Sequencing Kit consists of seven well characterized, highly pure, recombinant exoglycosidase enzymes selected to simplify the process of characterizing typical N-linked glycan structures. The extreme diversity of glycan structures on a glycoprotein makes elucidation of the profile challenging and often a number of orthogonal approaches are employed to verify the individual structures. The use of sequential or tandem exoglycosidase digestion of oligosaccharides followed by mass spectrometry or capillary electrophoresis analysis provides detailed carbohydrate sequence information and resolves ambiguities. The exoglycosidases are all active in a universal buffer system, GlycoBuffer 1, and can be used in single digests or in combination.
This kit is compatible with N-linked glycans released from a variety of CHO and murine derived antibodies, as well as N-linked glycans released from other glycoproteins. Optimal incubation times and enzyme concentration may vary for more complex glycans. The recommended enzyme quantity is sufficient for digestion of up to 130 pmol of released glycans (equivalent to released glycans from approximately 10 µgs of antibody) labeled with 2AA, 2AB or procainamide. Other commercially available “Instant labels” may require a larger quantity of enzyme or a longer incubation time for complete digestion.
N-glycan sequencing of antibodies
The enzymes provided are all active in a universal buffer system, GlycoBuffer 1, and can be used in single digests or in any combination.
All reagents supplied are mass spectrometry compatible.
Rapid PNGase F
Rapid™ PNGase F (non-reducing format)
PNGase F (Glycerol-free), Recombinant
α2-3,6,8,9 Neuraminidase A
α2-3 Neuraminidase S
β1-4 Galactosidase S
操作说明、说明书 & 用法
Protocols for E0577
Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
FAQs & 问题解决指南
Is the N-Glycan Sequencing Kit protocol suitable for digestion of glycoproteins?
How do I calculate the molarity of released glycans from my antibody substrate?
Why are two different neuraminidase enzymes provided with the N-Glycan Sequencing Kit?
Should I use the one- or two-step Rapid PNGase F deglycosylation method for my antibody?
The deglycosylation protocol included with the N-Glycan Sequencing Kit is recommended for up to 100 µg of antibody; How do I determine the amount of starting material I should deglycosylate?
Is it possible to combine the α1-2,3,6 Mannosidase with other exoglycosidases? Are any of the exoglycosidases inhibited by the Zinc that is required for optimal mannosidase activity?
Does ammonium formate inhibit exoglycosidase digestion?
How long do I need to incubate a Rapifluor-labelled substrate with the exoglycosidases?
Which is the enzyme of choice to release α1-6 fucose residues from antibody glycans: α1-2,4,6 Fucosidase O or α1-2,3,4,6 Fucosidase from Bovine Kidney?
Remove all traces of Acetonitrile before digesting labeled substrate with exoglycosidases for optimal results.
Use PCR tubes to avoid evaporation when incubating reactions overnight.
Include ZnCl2 in reactions with α1-2,3,6 Mannosidase (P0768) for optimal digestion.
Fucosidase O (P0749) requires an 18 hr incubation for complete digest of some instant labelled glycans and complex oligosaccharides with multiple branches.
If using Fucosidase O (P0749) alone, incubation temperature can be increased to 50°C for more efficient digestion.
引用 & 技术文献
Glycoproteomics Technical Guide
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Guthrie, E.P., Magneilli, P.E. (2016) Using glycosidases to remove, trim, or modify glycans on therapeutic proteins BioProcess Int; 14(2), 32-37.
Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein
Redesigning glycosidase manufacturing quality for pharmaceutical and clinical applications