New England Biolabs, P0741S, Endo S

Endo S is an endoglycosidase specific for cleaving the N-linked glycans from the chitobiose core of the heavy chain of native IgG Related Categories Endoglycosidases,, Proteome Analysis Applications Biotherapeutics and Antibody Analysis,, Expression Systems,, Glycan Sequencing, Specification Unit Definition One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 5 μg of native mouse monoclonal IgG in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 1 Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 55°C for 10 minutes Molecular Weight Apparent: 136 kDa Unit Assay Conditions 5 µg of IgG in 1X GlycoBuffer 1 are incubated with two-fold dilutions of Endo S for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE. FAQ Q: Is Endo S tagged? A: Endo S is tagged with a chitin binding domain (CBD). The CBD tag allows for removal from a reaction using chitin beads (NEB #S6651) or chitin magnetic beads (NEB #E8036). Note: there is no cleavage site between the Endo S and the CBD tag to enable removal of this tag from the Endo S. Q: What is the difference between PNGase F and Endo S? A: PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. Whereas, Endo S has a high specificity for removing N-linked glycans within the chitobiose core of native IgG. Q: How much Endo S should I use to deglycosylate a glycoprotein under native conditions? A: When working with native IgG, we recommend using 1 µL of Endo S per 100µg of native IgG. When working with other various glycoproteins (such as Fetuin) we recommend using a modified denaturing protocol, detailed below. Combine 100 µg of glycoprotein, 1 µL of 400mM DTT and H2O (if necessary to make a 10 µL total reaction volume. Denature glycoprotein by heating reaction at 55°C for 10 minutes. Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 1, H2O and 1-2 µL Endo S. Incubate reaction at 37°C for 1 hour. Note: To deglycosylate various glycoproteins, longer incubation times as well as more enzyme may be required. Reactions may be scaled-up linearly to accommodate larger reaction volumes. Q: What is the preferred substrate for Endo S? A: IgG is the preferred substrate. In addition, literature reports have shown that Endo S does not cleave human serum IgM or IgA * * Collin, M., Olsen, A. EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. The EMBO Journal (2001) Vol.20 No.12 pp.3046-3055. Q: What are the typical reaction conditions for Endo S? A: Typical reaction conditions are as follows: Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Combine 100 µg of native IgG, 1 µL of 10X GlycoBuffer 1 and H2O (if necessary) to make a 10 µL total reaction volume. Add 1 µL Endo S. Incubate reaction at 37°C for 1 hour. Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes. Q: How do I eliminate Endo S from a reaction? A: Endo S can be removed from a deglycosylation reaction using NEB’s magnetic chitin beads. Typical reaction conditions for removing 1-5 µL of Remove-iT® Endo S using magnetic chitin beads are as follows : Materials: Endo S (NEB #P0741) Chitin Magnetic Beads (NEB #E8036) Magnetic Separation Rack (NEB #S1506 or NEB #S1509) Pipette 50 µl Chitin Magnetic Beads into an eppendorf tube and place the eppendorf in a magnetic separation rack. Let the magnet attract the chitin beads, then pipette off the liquid supernatant and discard. With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 2 x 500 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant and discard. Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads. Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C. Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads. Pipette off the supernatant and keep. Wash the magnetic chitin beads 3 x 100 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep. Combine all supernatants from steps 5 & 6, as these are the deglycosylated glycoprotein. Analyze sample by method of choice. Note - Removal of Endo S from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes. Q: What is the binding capacity of the Magnetic Chitin Beads used to remove Endo S? A: The Magnetic Chitin Beads (NEB# E8036) binding capacity is approximately 0.4mg/ml of CBD-tagged protein. This binding capacity is calculated in mg of protein per bed volume of resin. The chitin magnetic beads are a 50:50 slurry. Therefore, 50 μl of slurry will yield 25 μl bed volume of resin, which is enought to remove 1-5 µL of Endo S. Q: Is Endo S compatible with downstream analysis such as HPLC and Mass Spectrometry? A: Endo S is supplied in a mass spec friendly, glycerol free storage buffer. Endo S can be used under native or DTT reaction conditions (see question 3), which are both compatible with downstream HPLC and MS analysis. 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.