New England Biolabs, P0711S, Rapid™ PNGase F (non-reducing format)

Related Categories Endoglycosidases,, Proteome Analysis Applications Biotherapeutics and Antibody Analysis,, Expression Systems,, Proteomics, Specification Reaction Conditions 1X Rapid PNGase F (non-reducing format) Buffer Incubate at 50°C Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 75°C for 10 minutes FAQ Q: What is the difference between Rapid PNGase F and Rapid PNGase F (non-reducing format)? A: Rapid PNGase F (non-reducing format) reaction conditions preserve the integrity of disulfide bonds, making it the reagent of choice for mass spectrometry studies of intact deglycosylated multimeric proteins (i.e. antibodies). Whereas, Rapid PNGase F (NEB #P0710) is ideal for downstream N-glycan analysis and profiling applications. In addition, Rapid PNGase F (non-reducing format) follows a 2-step protocol, as it has a strict requirement for a pre-incubation at 75°C. Q: What is the difference between Rapid PNGase F (non-reducing format) and Endo S (Endo S, NEB# P0741)? A: Rapid PNGase F (non-reducing format) 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. With Endo S, a single GlcNAc, with a fucose residue if present, remains attached to the asparagine residue of the IgG chain. Q: What are the components of Rapid PNGase F (non-reducing format)? A: Rapid PNGase F (non-reducing format) is provided with a proprietary 5X Rapid PNGase F (non-reducing format) Buffer (NEB# B0717S) for rapid deglycosylation under non-reducing conditions. Q: How much antibody sample can be deglycosylated with Rapid PNGase F (non-reducing format)? A: 1 µL of Rapid PNGase F (non-reducing format) in a 10 μL total reaction volume is optimal for rapid deglycosylation of up to 10 μg of antibodies containing both Fc and Fab glycosylation (such as Cetuximab), 15 μg of a mouse monoclonal antibody isotype IgG2a, and 30 μg of less complex antibodies, such as Rituximab. Reactions may be scaled-up linearly to accommodate larger amounts of antibody and/or glycoprotein. 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. Q: What should I do if my target protein is only partially deglycosylated with Rapid PNGase F (non-reducing format)? A: The amount of Rapid PNGase F (non-reducing format) Buffer can be increased up to 4 µl in a 10 µl total volume to facilitate glycan removal in complex substrates. For some proteins (for instance, Fetuin or chorionic gonadotropin) reducing agents are strictly required and complete rapid deglycosylation under non-reducing conditions will not be feasible. In such cases, we recommend the use of Rapid PNGase F (NEB# P0710). Q: What buffers or additives should be avoided when using Rapid PNGase F (non-reducing format)? A: The target protein should be in a solution compatible with Rapid PNGase F (non-reducing format) activity. Avoid buffers containing SDS, as it inhibits all versions of 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 if the Rapid PNGase F (non-reducing format) 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 actual sample to compare the position on a gel, and determine whether deglycosylation is partial or complete (refer to the “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. Q: Is Rapid PNGase F (non-reducing format) compatible with downstream mass spectrometry analysis? A: Yes, to prepare samples for mass spectrometry, the target protein can be prepared using micro-dialysis or micro-filtration (refer to “Glycoproteomics: Buffer Exchange Protocols”). Q: Is Rapid PNGase F (non-reducing format) 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 (non-reducing format) 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 (non-reducing format) in 10 minutes. Q: Are longer incubation times detrimental to the Rapid PNGase F (non-reducing format) 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 (non-reducing format)? A: Using the Rapid PNGase F Antibody Standard (NEB #P6043), which is a murine anti-MBP monoclonal antibody, isotype IgG2a, 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 (non-reducing format) 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: Can a protease inhibitor cocktail be used in a Rapid PNGase F (non-reducing format) reaction? A: Yes. Protease cocktails are compatible with the Rapid PNGase F (non-reducing format) 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: Is the Rapid PNGase F (non-reducing format) reaction compatible with Trypsin-ultra™ (NEB# P8101)? A: Yes, Trypsin-ultra™ Mass Spectrometry Grade (NEB# P8101) can be added directly to samples digested with Rapid PNGase F (non-reducing format). Protocol: To a 10ul Rapid PNGase F (non-reducing format) reaction containing 10ug of antibody, add 200 ug of Trypsin-ultra™ Mass Spectrometry Grade (NEB# P8101) for a trypsin/antibody ratio of 1:50. Incubate at 37°C for 2hrs (note: different substrates may require different amounts of trypsin, and optimized incubation times) Hydrophilic extraction methods are recommended to isolate peptides after a Rapid PNGase F (non-reducing format)/Trypsin reaction.