Endoglycosidase F1

References:

1. 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).

2. 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).

3. Reddy A., B. G. Grimwood, T. H. Plummer Jr and A. L. Tarentino. High- level expression of the Endo-beta-N- acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. Glycobiology 8:633-636 (1998).

4. Tarentino, A. L., C. M. Gomez and T. H. Plummer Jr. Deglycosylation of Asparagine-Linked Glycans by Peptide:N-Glycosidase F. Biochemistry 24:4665-4671 (1985).

5. Tarentino A. L., G. Quinones, W. P. Schrader, L. M. Changchien and T. H. Plummer Jr. Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem 267:3868-3872 (1992).

6. Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13):9702-9708 (1993).

7. 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).

8. Tarentino, A. L. and T. H. Plummer Jr. Enzymatic deglycosylation of asparagine- linked glycans: purification, properties and specificity of oligosaccharide- cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology 230:44-57 (1994).

9. Tarentino A. L., G. Quinones and T. H. Plummer Jr. Overexpression and purification of non-glycosylated recombinant endo-beta-N- acetylglucosaminidase F3. Glycobiology 5:599-601 (1995).

10. Trimble, R. B. and A. L. Tarentino. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biol Chem 266:1646-1651 (1991).

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Product Specification

Source: Recombinant Elizabethkingia miricola in E. Coli

EC: 3.2.1.96

Alternative Names: Endo F1, Endoglycosidase F1, endo-β-N-acetylglucosaminidase F

Endo F1 Specificity: Cleaves all asparagine-linked high mannose and some hybrid oligosaccharides

Contents:

60 µL aliquot of enzyme (1 U) in 20 mM Tris-HCl, pH 7.5
5x Reaction Buffer – 250 mM sodium phosphate, pH 5.5

Recommended Reagents
included with E-EF01 and E-EF01-20: 1 vial: 5x Reaction Buffer, 250 mM sodium phosphate, pH5.5

Activity ≥ 17 U/ml

Specific Activity ≥ 16 U/mg

Molecular Weight: 32 kD

Specific Activity : Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (RNase B) in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).

Formulation: The enzyme is provided as a sterile-filtered solution in 20mM Tris-HCl, pH 7.5

Storage: Store enzyme at 4°C. Do not freeze.

Stability: Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.

Specificity
Ludger Endo F1 cleaves Asparagine-linked high mannose or hybrid oligosaccharides. It cleaves 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. In contrast, PNGase F removes the oligosaccharide intact.

Quality & Purity
Ludger Endo F1 is 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 production host strain has been extensively tested and does not produce any detectable glycosidases.

Directions for use

1. Add up to 200 μg of glycoprotein to an Eppendorf tube.
2. Adjust to 38 μl final volume with de-ionized water.Add 10 μl 5x Reaction Buffer 5.
3. Add 2.0 μl of Endo F1 to the reaction. Incubate 1 hour or more at 37°C.
4. Monitor cleavage by SDS-PAGE.