New England Biolabs, P0702S, Endo H

Endoglycosidase H is a recombinant glycosidase which cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins. A tagged version, which is a recombinant protein fusion of Endoglycosidase H and maltose binding protein is also available, Endo H 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 (10 NEB units = 1 IUB milliunit). Unit Definition Assay: 10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of 1X GlycoBuffer 3, two-fold dilutions of Endo H are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE. 1X Glycoprotein Denaturing Buffer 0.5% SDS 40 mM DTT 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 75°C for 10 minutes Molecular Weight Apparent: 29 kDa FAQ 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: What is the difference between Endo H and Endo Hf? A: Endo H and Endo Hf are the same enzyme, but Endo Hf is the fusion protein of Endo H and MBP. The clone was engineered with the MBP attached to help in the purification of the enzyme. The fusion protein has identical activity to the non-fusion clone. There is no difference in activity between Endo H and Endo Hf on a glycoprotein. Q: I tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem? A: First, do you know how the carbohydrate is attached to the protein? Is it N-or O-linked (linked to an Asp or Ser/Thr)? Second, is the carbohydrate substrate high mannose type? If you are uncertain a more general endoglycosidase, PNGase F, may be the more appropriate enzyme. If you are certain that there are high mannose, N-linked carbohydrates, make sure that you have denatured the protein prior to deglycosylation. The secondary and tertiary structure of proteins often prevents 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 incubating longer. Remember, Endo H/Hf specifically removes only high mannose and some ""hybrid-type"" carbohydrates from glycoproteins. If the carbohydrate is more complex, then Endo H will not cleave. PNGase F is useful in removing ""all"" types of N-linked carbohydrates from glycoproteins. Q: How much Endo H/Endo Hf should I use? A: 1-2 ul of Endo H/Hf is enough to deglycosylate 5-20 μgs of denatured glycoprotein in one hour. Q: Is EndoH/ Endo Hf inhibited by SDS? A: No. Q: What are the typical reaction conditions for Endo H? 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 of 10X Glyco Buffer 3, H2O and 1-5 µl Endo H. 4. Incubate reaction at 37°C for 1 hour. Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes. Q: Are Protease Inhibitors acceptable for use in an Endo H/Hf reaction? A: 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. 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 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. 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.