|
The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide, or to use each enzyme independently and thereby determine the type of N-glycans present.
Product Description The Endo F Multi-kit is recommended to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions due to the glycan location within the protein’s three-dimensional structure, as these enzymes are known to be less sensitive to protein conformation.
Each of the enzymes has a different N-linked glycan specificity: Endoglycosidase F1 cleaves high mannose and some hybrid type N-glycans Endoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate) Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycans
Contents 1 vial: Endo F1- 20 μl (0.3 U) 20 mM Tris-HCl pH 7.5 1 vial: Endo F2- 20 μl (0.1 U) 10 mM sodium acetate, 25 mM NaCl, pH 4.5 1 vial: Endo F3- 20 μl (0.1 U) 20 mM Tris-HCl pH 7.5 1 vial: 5x Reaction Buffer - 400 μl 250 mM sodium acetate, pH4.5 1 vial: 5x Reaction Buffer - 400 μl 250 mM sodium phosphate, pH5.5
Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micro-mole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37°C, pH 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE.
Formulation The enzymes are provided as a sterile-filtered solution.
Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.
Specificity Endo F1 cleaves Asparagine-linked (N-linked) high mannose or hybrid oligosaccharides. Endo F2 cleaves N-linked biantennary oligosaccharides and high mannose (at a 40X reduced rate). Endo F3 cleaves free or N-linked fucosylated biantennary or triantennary oligosaccharides,as well as triamannosylchitobiose core structures. These enzymes cleave between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The recombinant version is not glycosylated, which may result in properties differing from the native protein.
Quality & Purity Endo F1, Endo F2, and Endo F3 are tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 34 µl final volume with de-ionized water. 2. Add 10 µl Endo F2 &F3 5x Reaction Buffer, 250 mM sodium acetate pH 4.5. Use Endo F1 buffer, 250 mM sodium phosphate pH 5.5 if you are using the Endo F1 enzyme alone. 4. Add 2.0 µl of each enzyme to the reaction. Incubate 3 hours at 37°C. Monitor cleavage by SDS-PAGE.
Applications – Deglycosylation of native proteins resistant to PNGase F cleavage – Determination of glycan type (high mannose, biantennary, tri/tetrantennary) – Deglycosylating proteins which normally precipitate when deglycosylating – X-Ray Crystallography
These three enzymes cleave asparagine-linked (N-linked) oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine, enhancing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact. |
