New England Biolabs, E0540S, O-Glycosidase & α2-3,6,8 Neuraminidase Bundle

This bundle contains 1 vial each of O-Glycosidase ( Related Categories Endoglycosidases Applications Expression Systems,, Glycan Sequencing,, Protein Digestion, Specification Unit Definition One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 μl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively). Non-denaturing Unit Definition of O-Glycosidase: Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of Neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent. Unit Definition of Neuraminidase: One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 μl. 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) FAQ Q: What are the typical reaction conditions for O-Glycosidase? A: Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows: Combine 10-20 µg of glycoprotein, 1 µL of 10X Glycoprotein Denaturing Buffer and H20 (if necessary) to make a 10 µL total reaction volume. Denature glycoprotein by heating reaction at 100°C for 10 minutes. Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 2, 2 µL 10% NP-40, 2 µL Neuraminidase, H20 and 1-5 µL O-Glycosidase. Incubate reaction at 37°C for 1 - 4 hours. Note: O-Glycosidase can be used under either denaturing or native conditions. However, under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent. The reaction may be scaled-up linearly to accommodate large amounts of O-Glycosidase and larger reaction volumes. Q: Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase? A: Yes. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (NEB #P0720) works well. Additionally, an O-Glycosidase and Neuraminidase Bundle (NEB# E0540S) is available. 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: How much O-Glycosidase should I use to remove my carbohydrate under native conditions? A: When the protein is not denatured O-Glycosidase 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: How do I inhibit O-Glycosidase? A: SDS will inhibit of O-Glycosidase. Q: What is a good positive control for O-Glycosidase? A: We suggest p-Nitrophenyl galacto-N-bioside (Galβ1-3GalNAcα1pNP, or Core1-pNP). Alternatively, fetuin (NEB #P6042S) can be digested with neuraminidase, and neuraminidase plus O-glycosidase. The changes in the molecular weight of fetuin after glycan removal can be observed by SDS-PAGE. 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: Do detergents inhibit O-Glycosidase? A: It is imperative to add NP-40 to a final concentration of 1% to your reaction mixture in order to counteract detergent inhibition. O-Glycosidase has full activity in the presence of 1% NP-40. Q: I tried using O-Glycosidase 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. O-Glycosidase will remove core 1 and core 3 O-linked disaccharides attached to Ser/Thr. If you are certain you have O-linked carbohydrates, make sure that you have treat your glycoprotein with Neuraminidase, and 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 O-Glycosidase from the SDS in the denaturation step).