New England Biolabs, P0720L, α2-3,6,8 Neuraminidase

Related Categories Exoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycan Sequencing,, Proteomics, Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 1 Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 50 mM NaCl 20 mM Tris-HCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 43 kDa Unit Assay Conditions Two fold dilutions of α2-3,6,8 Neuraminidase are incubated with 1 nmol AMC-labeled substrate and 1X GlycoBuffer 1 in a 10 μl reaction. The reaction mix is incubated at 37°C for 5 minutes. Separation of reaction products are visualized via thin layer chromatography (3). FAQ Q: Can I use α2-3,6,8 Neuraminidase in a double digest with Endo H(Hf) or PNGase F? A: Yes. As with the above question we've observed excellent α2-3,6,8 Neuraminidase activity at higher and lower pH's, and we've also found it to work very well on a protein that has been denatured. Neuraminidase shows normal activity even in the presence of 1% BME, and 0.5% SDS. Q: Is α2-3,6,8 Neuraminidase active at higher pH levels? A: Yes. α2-3,6,8 Neuraminidase has a very broad pH curve. Although optimum pH is around 5.5, we see excellent sialic acid removal at pH's as low as 4.5 and as high as pH 8.5. We have used α2-3,6,8 Neuraminidase at pH ranges more suitable for PNGase F (pH range 7.5-8.6). Q: How much exoglycosidase should be used? A: The amount of enzyme required varies when different substrates are used. Start with 1-2 µl for 1µg of glycoprotein or 100 nM of oligosaccharide for one hour in a 10-25 µl reaction. If there is still undigested material, let the reaction go overnight. Q: Do detergents inhibit exoglycosidases/endoglycosidases? A: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. Exceptions are: PNGase F (all formulations), O-glycosidase, and β-N-Acetylglucosaminidase, which are inhibited by SDS. It is imperative to add NP-40 to a final concentration of 1% to your reaction mixture in order to counteract detergent inhibition. Endo D is also inhibited by SDS, however NP-40 does not counteract this inhibition. Finally, common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaines, as well as traces of organic solvents, can prevent optimal rapid deglycosylation by Rapid PNGase F. Q: What is a good positive control for α2-3,6,8 Neuraminidase? A: We suggest Fetuin (NEB #P6042). The changes in the molecular weight of fetuin after glycan removal can be observed by SDS-PAGE. Alternatively, the chromogenic substrate pNP-N-acetyl-neuraminic acid can be used for molecular weight visualization. Q: Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers? A: To simplify workflows and digestions with two or more exoglycosidases, most enzymes are now provided with 10X GlycoBuffer 1. Two exceptions are provided with GlycoBuffer 4. Also, the buffer panel for endoglycosidases has been simplified. The activity of every endo- and exoglycosidase enzyme was evaluated in both its old and new buffer system. All enzymes retain either the same activity, or were found to have greater activity in the new buffer. Some exoglycosidases are still provided with an additional vial of rHSA (rHSA enhances the activity of certain enzymes). As before, some exoglycosidases are still provided with an additional vial of BSA (BSA enhances the activity of certain enzymes). Other components (i.e. Glycoprotein Denaturing Buffer, NP-40, etc.) are still provided when required. Enzyme Product # Old buffer Current buffer Changed? α-N-Acetylgalactosaminidase P0734 G7 GlycoBuffer 1 YES α1-2 Fucosidase P0724 G4 GlycoBuffer 1 YES α1-2,3,4,6 Fucosidase P0748 --- GlycoBuffer 1 --- α1-2,4,6 Fucosidase O p0749 --- GlycoBuffer 1 --- α1-3,4 Fucosidase p0769 --- GlycoBuffer 1 --- α1-2,3 Mannosidase P0729 G6 GlycoBuffer 1 no α1-6 Mannosidase P0727 G2 GlycoBuffer 1 YES α1-2,3,6 Mannosidase p0768 --- GlycoBuffer 4 --- α1-3,4,6 Galactosidase P0747 --- GlycoBuffer 1 ---- α1-3,6 Galactosidase P0731 G6 GlycoBuffer 1 no α2-3 Neuraminidase S P0743 --- GlycoBuffer 1 --- α2-3,6,8 Neuraminidase P0720 G1 GlycoBuffer 1 YES α2-3,6,8,9 Neuraminidase A P0722 --- GlycoBuffer 1 --- β-N-Acetylhexosaminidasef P0721 G2 GlycoBuffer 1 YES β-N-Acetylglucosaminidase P0732 G1 GlycoBuffer 1 YES β-N-Acetylglucosaminidase S P0744 --- GlycoBuffer 1 --- β1-3 Galactosidase P0726 G6 GlycoBuffer 1 no β1-4 Galactosidase S P0745 --- GlycoBuffer 1 --- β1-3,4 Galactosidase p0746 --- GlycoBuffer 4 --- N-Glycan Sequencing Kit E0577 --- GlycoBuffer 1 --- Endo S P0741 G6 GlycoBuffer 1 YES Endo H P0702 G5 GlycoBuffer 3 YES Endo Hf P0703 G5 GlycoBuffer 3 YES Endo F2 p0772 --- GlycoBuffer 4 --- Endo F3 p0771 --- GlycoBuffer 4 --- Endo D P0742 G7 GlycoBuffer 2 no O-Glycosidase P0733 G7 GlycoBuffer 2 no O-Glycosidase & Neuraminidase Bundle E0540 G7 GlycoBuffer 2 no Remove-iT® PNGase F P0706 G7 GlycoBuffer 2 no PNGase F P0704 G7 GlycoBuffer 2 no PNGase F (Glycerol-free) P0705 G7 GlycoBuffer 2 no PNGase F, Recombinant P0708 G7 GlycoBuffer 2 no PNGase F (Glycerol-free), Recombinant P0709 G7 GlycoBuffer 2 no PNGase A p0707 --- Glyco Buffer 3 --- Protein Deglycosylation Mix II p6044 --- Deglycosylation Mix Buffer 1 and 2 --- Rapid PNGase F P0710 --- Rapid PNGase F Buffer ---- Rapid™ PNGase F (non-reducing format) p0711 --- Rapid PNGase F (non-reducing format) Buffer Q: What are Glycosidases and their uses? A: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the non-reduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography. Q: Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase? A: Yes. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (NEB #P0720) works well. Additionally, an O-Glycosidase and Neuraminidase Bundle (NEB# E0540S) is available. Q: Do the NEB Neuraminidase enzymes cleave both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) residues? A: Depending on the enzyme source and specificity, some neuraminidases cleave both Neu5Ac and Neu5Gc, while some cleave only Neu5Ac. Those that cleave both Neu5Ac and Neu5Gc, tend to have lower efficiency towards Neu5Gc. NEB# P0720 (a2-3,6,8 Neuraminidase) – cleaves both Neu5Ac and Neu5Gc NEB# P0722 (a2-3,6,8,9 Neuraminidase A) – cleaves both Neu5Ac and Neu5Gc NEB# P0743 (a2-3 Neuraminidase S) – cleaves only Neu5Ac