New England Biolabs, P0772S, Endo F2

Related Categories Endoglycosidases Applications Glycan Sequencing Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the carbohydrate from 10 µg Porcine Fibrinogen in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 4 Incubate at 37°C 1X GlycoBuffer 4 50 mM sodium acetate (pH 4.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 39.8 kDa Unit Assay Conditions Two fold dilutions of Endo F2 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. FAQ Q: Is Endo F2 tagged? A: Endo F2 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 F2 and the CBD tag to enable removal of this tag from the Endo F2. Q: What is the difference between PNGase F and Endo F2? A: PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. Whereas, Endo F2 has a high specificity for removing N-linked glycans within the chitobiose core of glycoproteins containing only biantennary and (at a reduced rate) high mannose oligosaccharides. Q: What is the difference between Endo F2 and Endo F3? A: Endo F2 removes N-linked glycans within the chitobiose core of glycoproteins containing only biantennary and (at a reduced rate) high mannose oligosaccharides. 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 F2 should I use to deglycosylate a glycoprotein under native conditions? A: We recommend using 1µl of Endo F2 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 F2 Incubate reaction at 37°C for 1 hour. Q: What is the preferred substrate for Endo F2? A: Porcine Fibrinogen is a glycoprotein that contains fucosylated-biantennary N-linked glycans and can be used as a positive control for Endo F2. Q: Do detergents inhibit Endo F2 activity? A: Endo F2 is typically used under non-denaturing (native) conditions, as denaturation is not necessary for optimal activity of the enzyme. However, Endo F2 is active in denaturing conditions. Concentrations of 0.5% SDS do not inhibit Endo F2 activity when used in the presence of 1% NP-40. Q: What is the optimal pH for Endo F2 activity? A: The optimal pH for cleavage of biantennary glycans by Endo F2 is pH 4.5 (10X Glycobuffer 4). Q: How do I eliminate Endo F2 from a reaction? A: Endo F2 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 F2 (NEB# P0772) 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, NEB# S1509) . 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 & 6, as these are the deglycosylated glycoprotein. Analyze sample by method of choice Note - Removal of Endo F2 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 F2? A: The Magnetic Chitin Beads (NEB# E8036) binding capacity is 0.4 µg/µl of CBD-tagged protein. Q: Is Endo F2 compatible with downstream analysis such as HPLC and Mass Spectrometry? A: Endo F2 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.