Endo F Multi-Kit
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.
Part Number: KE-EFX3
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
– 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.
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.
Endo F References:
Maley P., R. B. Trimble, A. L. Tarentino and T. H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180:195-204 (1989).
Plummer, T. H. Jr, A. W. Phelan and A. L. Tarentino. Porcine fibrinogen glycopeptides: substrates for detecting endo-N-acetylglucosaminidases F2 and F3. Anal Biochem 235:98-101 (1996).
Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidaseF activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13):9702-9708 (1993).
Tarentino A. L. and T. H. Plummer Jr. Substrate specificity of Flavobacterium meningosepticum: Endo F2 and endo F3: purity is the name of the game. Glycobiology 4:771-773 (1994).