New England Biolabs, P0704S, PNGase F

PNGase F is the most effective enzymatic method for removing almost all Related Categories Endoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycan Sequencing,, Proteomics, Specification Unit Definition One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl. Unit Definition Assay: 10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. 1X Glycoprotein Denaturing Buffer 0.5% SDS 40 mM DTT 1X NP-40 1% NP-40 in MilliQ-H 2 O Reaction Conditions 1X GlycoBuffer 2 Incubate at 37°C 1X GlycoBuffer 2 50 mM Sodium Phosphate (pH 7.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 75°C for 10 minutes Molecular Weight Apparent: 36000 daltons FAQ Q: Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry? A: NEB sells two versions of the enzyme, PNGase F (NEB #P0704) and PNGase F (Glycerol-free) (NEB #P0705). The two versions of the enzyme have identical activity, specificity and concentration. The only difference between the two is that PNGase F is stored in 50% Glycerol, while PNGase F (Glycerol-free) does not contain glycerol in its storage buffer. PNGase F (Glycerol-free) is recommended when downstream analysis will include 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, we recommend using PNGase F (Glycerol-free) under non-denaturing conditions if HPLC or MS will be used for downstream analysis. Non-denaturing conditions often require more enzyme units and a longer incubation time. Q: What happens to the asparagine after PNGase removes the sugar? A: Since the enzyme is a glycoamidase, the asparagine gets converted into aspartic acid. Q: Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein? 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 F 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 the typical reaction conditions for PNGase F? A: Typical reaction conditions are as follows: 1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume. 2. Denature glycoprotein by heating reaction at 100°C for 10 minutes. 3. Make a total reaction volume of 20 µl by adding 2 µl 10X GlycoBuffer 2, 2 µl 10% NP40, H2O and 1-2 µl PNGaseF. 4. Incubate reaction at 37°C for 1 hour. Note: We recommend limiting PNGaseF to 1/10 (or less) of the total reaction volume to keep final glycerol concentration equal to (or less than) 5%. Reaction may be scaled-up linearly to accommodate large amounts of PNGaseF and larger reaction volumes. Q: Does PNGase F work in Urea? A: PNGase F is stable in 2.5M urea at 37C for 24 h and still possesses 40% of its activity in 5 M urea1. 1. Maley, F., et al, Anal Biochem, 180, 195-204 (1989) Q: How do I inhibit PNGase F? A: SDS is an excellent inhibitor of PNGase F. Q: How much PNGase F should I use to remove my carbohydrate under native conditions? A: When the protein is not denatured PNGase F can have a difficult time reaching the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Sometimes extra enzyme and extended incubation times can help but these values are specific to each protein and must be determined empirically. Q: I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem? A: Do you know how the carbohydrate is attached to the protein? Is it N-, or O-linked (linked to an Asn, or a Ser/Thr)? PNGase F will only remove carbohydrates attached to an Asn. NEB’s O-Glycosidase (NEB# P0733) will remove core 1 and core 3 O-linked carbohydrates attached to Ser/Thr. If you are certain you have N-linked carbohydrates, make sure that you denature the protein prior to deglycosylation. The secondary and tertiary structure of proteins can prevent endoglycosidases from reaching their substrate, thus making denaturation a crucial step in efficient cleavage. If you do not want to denature then consider adding more enzyme and longer incubation times. If you have denatured the protein, be certain to add NP-40 (to protect PNGase F from the SDS in the denaturation step). Outside these reaction conditions there is the possibility that the carbohydrate may be resistant to PNGase F (a rare occurrence). This happens when the core N-acetyl-glucosamine is modified by an α1-3Fucose (often found in plant proteins). Q: What is the difference between PNGase F, Endo H and O-Glycosidase? A: PNGase F removes all types of N-linked (Asn linked) glycosylation; high mannose, hybrid, bi, tri, and tetra-antennary. You will choose this enzyme if your goal is to remove all N- linked carbohydrates without regard to type. Endo H removes only high mannose and some hybrid types of N-linked carbohydrates. You would choose this enzyme to more closely determine the type of N-linked glycosylation, or if you know that the protein has a carbohydrate sensitive to Endo H. NEB’s O-Glycosidase will remove desialyated core 1 and core 3 O-linked disaccharides attached to Ser/Thr residues. 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: 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: What is a good endoglycosidase substrate? A: For PNGaseF and Protein Deglycosylation Mix we suggest Fetuin (NEB #P6042, supplied with the Protein Deglycosylation Mix). For PNGaseF, Endo H, and Endo Hf, we suggest RNase B (NEB #P7817). For Endo D we suggest Phospholipase A from bee venom. For Endo S we suggest mouse monoclonal IgG (NEB #E8032). For O-glycosidase we suggest p-Nitrophenyl galacto-N-bioside (Galβ1-3GalNAcα1pNP, or Core1-pNP). Alternatively, fetuin can be digested with neuraminidase, and neuraminidase plus O-glycosidase. The changes in molecular weight after glycan removal can be observed by SDS-PAGE.