New England Biolabs, P0707S, PNGase A

PNGase A cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and short complex oligosaccharides such as those found in plant and insect cells from N-linked glycoproteins and glycopeptides. PNGase A differs from PNGase F in that it cleaves N-linked glycans with or without α(1,3)-linked core fucose residues. Related Categories Endoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycobiology & Proteomics,, Proteomics, Specification Unit Definition One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 1 µg of denatured recombinant Avidin produced in Maize in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 3 Incubate at 37°C 1X GlycoBuffer 3 50 mM sodium acetate (pH 6 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 63.8 kDa Unit Assay Conditions 1 μg of recombinant Avidin is denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 3, two-fold dilutions of PNGase A are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. FAQ Q: What is the difference between PNGase F and PNGase A? A: PNGase F and PNGase A both cleave between the innermost GlcNAc and asparagine residues of N-linked glycans on both glycoproteins and glycopeptides. PNGase F can cleave almost all N-linked glycans from high mannose, hybrid, and complex oligosaccharides. However, PNGase F cannot cleave N-glycans with core α1-3 fucosylation. In contrast, PNGase A cleaves N-linked glycans from high mannose, hybrid, and short complex oligosaccharides such as those found in plant and insect cells. PNGase A differs from PNGase F in that it cleaves N-linked glycans with or without α(1,3)-linked core fucose residues. Q: Can PNGase A be used under non-denaturing (native) conditions? A: No, PNGase A requires that the glycoprotein be at least mildly reduced prior to deglycosylation. We recommend using PNGase A with only DTT at a final concentration of 40 mM. The reaction should be heated at 55°C for 10 minutes and then cooled before adding the PNGase A. Increasing the amount of PNGase A or the length of incubation at 37°C may be necessary to achieve full deglycosylation. Q: Which high mannose structures can PNGase A cleave? A: PNGase A is able to cleave high mannose N-glycan structures from Man 3 up to Man 9. Q: Can PNGase A cleave large, complex oligosaccharides? A: PNGase A cannot cleave larger N-glycans such as those from Fetuin, Fibrinogen, IgG, Lactoferrin and Transferrin. Q: What happens to the asparagine after PNGase A removes the sugar? A: Since the enzyme is a glycoamidase, the asparagine is converted into aspartic acid. Q: What is a good PNGase A substrate? A: NEB’s PNGase A will cleave both glycoproteins and glycopeptides. We suggest using recombinant Avidin from maize, or horseradish peroxidase (HRP) as positive controls. Q: Is PNGase A compatible with downstream analysis such as HPLC and Mass Spectrometry? A: PNGase A is supplied in a glycerol-free storage buffer to allow for compatibility with downstream analysis such as HPLC and/or Mass Spectrometry due to the fact that glycerol is not tolerated in such instruments. Glycoprotein Denaturing Buffer (containing SDS and DTT) is not compatible with Mass Spectrometry applications, and often times hard to remove from the reaction. Therefore, to create a mass spectrometry compatible reaction, we recommend using PNGase A with only DTT at a final concentration of 40 mM. The reaction should be heated at 55°C for 10 minutes and then cooled before adding the PNGase A. Increasing the amount of PNGase A or the length of incubation at 37°C may be necessary to achieve full deglycosylation. Q: How much PNGase A should I use to remove my carbohydrate under native or DTT denaturing conditions? A: When the protein is not denatured with SDS, PNGase A has to work harder to reach the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Sometimes additional enzyme and extended incubation times can help but these values are specific to each protein and must be determined empirically. Q: Why is my protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein. Can a protease inhibitor cocktail be used in a PNGase A reaction? A: When a protein is denatured it is more susceptible to cleavage by proteases. For this reason a protease cocktail containing the following can be used in a PNGase A reaction protocol: Aprotinin (10 μg/ml final concentration) Benzamidine (1mM final concentration) Pepstatin (10 μg/ml final concentration) Leupeptin (1μM final concentration) EGTA (1 mM final concentration) EDTA (1 mM final concentration) PMSF (1 mM final concentration) Make a 1000X concentrated stock of each inhibitor in water; except Pepstatin which should be dissolved in methanol. Note: PMSF has the ability to modify basic residues on glycoprotein substrates. 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.