New England Biolabs, P0710S, Rapid™ PNGase F

Rapid PNGase F enables complete and rapid deglycosylation of antibodies and immunoglobulin fusion proteins, as well as other glycoproteins, in minutes. All Related Categories Endoglycosidases,, Proteome Analysis Applications Biotherapeutics and Antibody Analysis,, Expression Systems,, Glycan Sequencing, Specification Reaction Conditions 1X Rapid PNGase F Buffer Incubate at 50°C Heat Inactivation 75°C for 10 minutes FAQ Q: What is the difference between Rapid PNGase F and Endo S (Endo S, NEB# P0741)? A: Rapid PNGase F cleaves N-glycans from antibodies between the innermost GlcNAc and asparagine residues. Endo S is an endoglycosidase that cleaves within the chitobiose core of N-linked glycans from the heavy chain of native IgG. A single GlcNAc, with a fucose residue if present, remains attached to the asparagine residue of the IgG chain. Q: What is the difference between Rapid PNGase F and PNGase F? A: Rapid PNGase F has been developed to deglycosylate antibodies and related fusion proteins in minutes. The convenient reaction set up and the fast reaction time makes Rapid PNGase F ideal for high throughput applications during development and quality control of therapeutic monoclonal antibodies. Q: What are the components of Rapid PNGase F? A: Rapid PNGase F is provided with its own proprietary 5x Rapid PNGase F Buffer. Q: What are the typical reaction conditions for Rapid PNGase F? A: For optimal heat transfer, use 0.2ml thin wall microtubes or alternatively, 1.5 ml centrifuge tubes. A thermal cycler with heated lid, or a microtube heat block, are suitable for incubation. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and/or larger reaction volumes. One step protocol: Combine up to 100 μg of antibody and H2O to a volume of 16 μl. Add 4 μl of 5X Rapid PNGase F Buffer to make a 20 μl total reaction volume. Add 1 μl of Rapid PNGase F. Incubate 10 minutes at 50°C. Prepare N-glycans for derivatization (i.e. reductive amination) for downstream analysis. To prepare a deglycosylated protein for mass spectrometry analysis, buffer exchange by micro-dialysis or micro-filtration. Refer to the “Glycoproteomics: Buffer Exchange Protocols” for more detail. Two step protocol: Some antibodies (i.e. Fab N-glycans) require a pre-heating step for efficient deglycosylation. Combine up to 100 μg of antibody and H2O to a volume of 16 μl. Add 4 μl of 5X Rapid PNGase F Buffer to make a 20 μl total reaction volume. Incubate at 80°C for 2 minutes, cool down. Add 1 μl of Rapid PNGase F. Incubate 10 minutes at 50°C. Prepare N-glycans for derivatization (i.e. reductive amination) for downstream analysis. To prepare a deglycosylated protein for mass spectrometry analysis, buffer exchange by micro-dialysis or micro-filtration. Refer to the “Glycoproteomics: Buffer Exchange Protocols” for more detail. Q: How do I know whether to follow the “One Step” or the “Two Step” protocol? A: Rapid PNGase F has been developed for efficient and fast deglycosylation of antibodies in a simple one step reaction at 50°C. However, some IgGs (i.e. carrying Fab glycosylation) require a pre-denaturing step of 2 minutes at 80°C. We recommend starting with the standard One Step protocol. If there is evidence (i.e. by gel migration or proteomic analysis) that N-glycans remain attached to the protein, follow the Two Step protocol. Q: How much Rapid PNGase F should I use to deglycosylate my antibody sample? A: The quantity of enzyme recommended is sufficient for the deglycosylation of up to 100 μg of a mouse monoclonal antibody isotype IgG2a. The optimal amount of starting material will be determined by the nature of a sample (glycan diversity) and the particular downstream N-glycan analysis that will be performed. Typically, 30-50 µg of antibody are sufficient for complete N-glycan quantitation (after labeling by reductive amination) by HILIC followed by ESI-MS. For proteins with additional glycan sites, and/or a high glycosylation variability, more starting material might be required to quantitate all minor glycan species. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and/or larger reaction volumes. Q: What buffers or additives should be avoided when using Rapid PNGase F? A: The target protein should be in a solution compatible with Rapid PNGase F activity. Avoid buffers containing SDS, as it inhibits PNGase F. 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. Q: How can I test whether the Rapid PNGase F reaction is complete in 10 minutes? A: For a monoclonal antibody, a SDS-PAGE shift can be observed after removal of N-glycans. Typically, this is only a modest change, so it is crucial to run a control along with the real sample to compare the position on a gel, and determine whether deglycosylation is partial or complete (refer to the “Analysis of a Rapid PNGase F Deglycosylation Reaction by SDS-PAGE” protocol). To detect whether or not minor amounts of glycosylated antibody remain, a more sensitive assay is required. For instance, a proteomic analysis by LS-ESI-TOF will show the different glycosylated and non-glycosylated forms present (refer to the “Intact Protein LS-ESI-TOF Protocol”). Q: Are downstream analysis such as HPLC and Mass Spectrometry compatible with Rapid PNGase F? A: Yes, to prepare samples for liquid chromatography and/or mass spectrometry, N-glycans can be isolated by conventional solid phase extraction methods such as C18 and graphitized carbon (PGC). Refer to the “Glycan SPE C18 and Graphitized Carbon Protocols”. In addition to C18 and PGC, hydrophilic interaction (HILIC) may also be used downstream. For a proteomics experiment, the target antibody can be prepared using micro-dialysis or micro-filtration (refer to the “Glycoproteomics: Buffer Exchange Protocols”). Q: Is Rapid PNGase F only suitable for deglycosylation of antibodies? A: Although this product has been optimized for the rapid removal of N-glycans from antibodies, it can be utilized with various other glycoproteins. Rapid PNGase F has been shown to act on other proteins such as fibrinogen and transferrin. However; some proteins tested, such as Fetuin, were not efficiently deglycosylated with Rapid PNGase F in 10 minutes. Q: Are longer incubation times detrimental to the Rapid PNGase F reaction? A: No. Identical results (quantitative and qualitative yields of N-glycans released as well as intact analysis of the deglycosylated antibody) were achieved following a 10 minute or 1 hour incubation period. Q: What is the reaction temperature range for Rapid PNGase F? A: Using a mouse monoclonal antibody, effective deglycosylation was observed in 10 minutes at temperature ranging from 48 to 58°C. The optimal temperature for the use of Rapid PNGase F is 50°C. Q: What happens to the asparagine residue after Rapid PNGase F removes the sugar? A: Since the enzyme is a glycoamidase, the asparagine residue is converted into aspartic acid. Q: Will a Rapid PNGase F reaction preserve the integrity of glycosylamines? A: No. Although N-glycans are released as glycosylamines by Rapid PNGase F, the amino group is unstable and it is rapidly converted. The deglycosylation reaction results in N-glycans with a free reducing end. Q: Can a protease inhibitor cocktail be used in a Rapid PNGase F reaction? A: Yes. Protease cocktails are compatible with the Rapid PNGase F reaction. The following protease inhibitors have been tested: 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) and PMSF (1 mM final concentration). Please note that we do not recommend PMSF as it has the ability to modify basic residues on glycoprotein substrates. Q: What is a good control substrate for Rapid PNGase F? A: Rapid PNGase F Antibody Standard, NEB #P6043. The Rapid PNGase F Antibody Standard is a murine anti-MBP monoclonal antibody, isotype IgG2a.