{"product_id":"neb-p0742s","title":"New England Biolabs, P0742S, Endo D","description":"The large size of this product was discontinued on June 15, 2025. The small size will remain available.  \u003cb\u003eRelated Categories\u003c\/b\u003e Endoglycosidases,, Proteome Analysis  \u003cb\u003eApplications\u003c\/b\u003e Expression Systems,, Glycan Sequencing,, Proteomics,  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e One unit is defined as the amount of enzyme required to remove \u0026gt; 95% of the carbohydrate from 10 μg of glycosidase-trimmed (trimannosyl core) Fetuin in 1 hour at 37°C in a total reaction volume of 10 μl.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X GlycoBuffer 2 Incubate at 37°C 1X GlycoBuffer 2 50 mM Sodium Phosphate (pH 7.5 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e 65°C for 10 minutes  \u003cb\u003eMolecular Weight\u003c\/b\u003e Apparent: 140 kDa  \u003cb\u003eUnit Assay Conditions\u003c\/b\u003e 10 μg of glycosidase-trimmed (trimannosyl core) Fetuin are denatured with 1X DTT at 95°C for 3 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of Endo D are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.  \u003cb\u003eFAQ\u003c\/b\u003e Q: What is the tag on Endo D? A: Endo D is tagged with a chitin binding domain (CBD). The CBD tag allows for removal from a reaction using magnetic chitin beads (NEB #E8036). Note: there is no cleavage site between the Endo D and the CBD tag to enable removal of this tag from the Endo D. Q: What is the difference between PNGase F, Endo S and Endo D? A: PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. Endo S has a high specificity for removing N-linked glycans within the chitobiose core of native IgG. Whereas, Endo D cleaves within the chitobiose core of paucimannose N-linked glycans from glycoproteins and glycopeptides, with or without extensions in the antennae. Q: What is a typical reaction protocol for Endo D? A: We recommend using 1 µL of Endo D per 20µg of paucimannose N-linked glycans. Combine 10-20 µg of glycoprotein, 1 µL of 10X DTT and H2O (if necessary to make a 10 µL total reaction volume. Denature glycoprotein by heating reaction at 95°C for 3 minutes. Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 2, H2O and 1 µL Endo D. Incubate reaction at 37°C for 1 hour. Note: To deglycosylate various glycoproteins under native conditions, longer incubation times as well as more enzyme may be required. Reactions may be scaled-up linearly to accommodate larger reaction volumes. Q: Will SDS inhibit Endo D? A: Yes, Endo D is inhibited by SDS and unlike other endoglycosidases, NP-40 does not counteract the SDS inhibition. Endo D is therefore not recommended for use with NEB’s Glycoprotein Denaturing Buffer which contains both SDS and DTT. The enzymeco is instead sold with a 10X DTT solution for denaturation purposes without SDS. Q: How do I eliminate Endo D from a reaction? A: Endo D can be removed from a deglycosylation reaction using NEB’s Magnetic Chitin Beads (NEB #E8036). Typical reaction conditions for removing 1-5 µL of Remove-iT® Endo D using magnetic chitin beads are as follows: Materials: Endo D (NEB #P0742), Chitin Magnetic Beads (NEB #E8036), Magnetic Separation Rack (NEB #S1506, 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 NH4Formate 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 NH4Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep. Combine all supernatants from steps 5 \u0026amp; 6, as these are the deglycosylated glycoprotein. Analyze sample by method of choice . Notes: Removal of Endo D from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes. The ideal reaction volume for 50 μl of chitin resin is in the range of equal volume to no more than 5X bead bed volume. Q: What is the binding capacity of the Magnetic Chitin Beads used to remove Endo D? A: The Magnetic Chitin Beads (NEB# E8036) Binding Capacity is approximately 0.4 mg\/ml 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 enough to remove 1-5 µL of Endo D. Q: Is Endo D compatible with downstream analysis such as HPLC and Mass Spectrometry? A: Endo D is supplied in a mass spec friendly, glycerol free storage buffer. Endo D 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 \u0026amp; 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: Can Endo D cleave glycoproteins under native conditions? A: Yes, Endo D is able to cleave glycans from some glycoproteins under native conditions if the glycan structure is trimmed to the trimannosyl core. For example, 10ugs of Rituximab (chimeric human\/mouse IgG1 antibody) was incubated with the following glycosidases: 50 units of a2-3,6,8,9 Neuraminidase A (NEB #P0722 ), 8 units of b1-4 Galactosidase S (NEB #P0745), 8 units of β-N-Acetylglucosaminidase S (NEB #P0744 ) and 250 units of Endo D (NEB #P0742 ) in 1X Glycobuffer 1 and incubated overnight at 37°C. This resulted in complete removal of the glycan from the antibody. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835495141545,"sku":"P0742S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-p0742s","provider":"Iright","version":"1.0","type":"link"}