{"product_id":"neb-p0771s","title":"New England Biolabs, P0771S, Endo F3","description":"Endo F3 is an endoglycosidase that cleaves within the chitobiose core of N-linked fucosylated-biantennary and triantennary complex oligosaccharides from glycoproteins. Endo F3 is tagged with chitin binding domain (CBD) for easy removal.  \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 cleave \u0026gt; 95% of the carbohydrate from 10 µg Porcine Fibrinogen in 1 hour at 37°C in a total reaction volume of 10 μl.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X GlycoBuffer 4 Incubate at 37°C GlycoBuffer 4 50 mM sodium acetate (pH 4.5 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 20 mM Tris-HCl 50 mM NaCl 5 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: 38.8 kDa  \u003cb\u003eUnit Assay Conditions\u003c\/b\u003e Two fold dilutions of Endo F3 are incubated with 10 µg Porcine Fibrinogen and 1X GlycoBuffer 4 in a 10 μl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via SDS-PAGE.  \u003cb\u003eFAQ\u003c\/b\u003e Q: Is Endo F3 tagged? A: Endo F3 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 F3 and the CBD tag to enable removal of this tag from the Endo F3. Q: What is the difference between PNGase F and Endo F3? A: PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. Whereas, Endo F3 has a high specificity for removing N-linked glycans within the chitobiose core of glycoproteins containing fucosylated-biantennary and triantennary oligosaccharides. Q: How much Endo F3 should I use to deglycosylate a glycoprotein under native conditions? A: Endo F3 should always be used under native conditions. We recommend using 1µl of Endo F3 per 20µg of native glycoprotein. Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows: Combine 20 µg of glycoprotein, 1 µL of 10X Glycobuffer 4 and H20 (if necessary) to make a 10 µL total reaction volume. Add 1 µL Endo F3 Incubate reaction at 37°C for 1 hour. Q: What is the preferred substrate for Endo F3? A: Porcine Fibrinogen is a glycoprotein that contains fucosylated-biantennary N-linked glycans and can be used as a positive control for Endo F3. Q: Do detergents inhibit Endo F3 activity? A: Endo F3 should only be used under non-denaturing (native) conditions. Detergents, such as SDS, inhibit Endo F3 activity. Q: What is the optimal pH for Endo F3 activity? A: The optimal pH for the cleavage of fucosylated-biantennary (i.e. porcine fibrinogen) and triantennary glycans (i.e. fetuin) by Endo F3 is pH 4.5 (10X Glycobuffer 4). However, fucosylated-biantennary glycans can also be cleaved by Endo F3 at pH 6.0 (10X Glycobuffer 3). Q: How do I eliminate Endo F3 from a reaction? A: Endo F3 can be removed from a deglycosylation reaction using NEB’s magnetic chitin beads. Typical reaction conditions using magnetic chitin beads are as follows: Materials: Endo F3 (NEB #P0771 ) Chitin Magnetic Beads (NEB #E8036) Magnetic Separation Rack (NEB #S1506, NEB #S1509) Pipette 50µl chitin magnetic beads ((NEB #E8036) into an eppendorf tube and place the eppendorf in a magnetic separation rack (NEB #S1506 or #S1509S). 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 3 x 500uL 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 100uL 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 Note - Removal of Endo F3 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 F3? A: The Magnetic Chitin Beads (NEB #E8036 ) binding capacity is 0.4 µg\/µl of CBD-tagged protein. Q: Is Endo F3 compatible with downstream analysis such as HPLC and Mass Spectrometry? A: Endo F3 and 10X Glycobuffer 4 are both mass spec and HPLC compatible. 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. 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