background:

Endoglycosidase F3 (Endo F3) cleaves between two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of oligosaccharides, generating truncated sugar molecules with one N-acetylglucosamine residue remaining on the asparagine. Endo F3 has no activity against oligomannose and hybrid molecules. Endo F3 cleaves nonfucosylated bi- and triangular complex oligosaccharides at a slower rate, but only on peptide-linked oligosaccharides. Core fucosylation of the bi-angular structures increases its activity up to 400-fold. Core-fucosylated bi-angular structures are efficient substrates for Endo F3, even in free oligosaccharides. Endo F3 also cleaves fucosylated trimannose core structures on free and protein-linked oligosaccharides. Native deglycosylation of complex tetragonal glycans requires sequential hydrolysis to the triglycined diacetylchitobiose (Man3GlcNAc2) core followed by cleavage by Endo F3.

Rhinogen ® Endo F3 is recombinantly expressed in E. coli with an MBP fusion protein at the N-terminus and a 6×His tag at the C-terminus. Rhinogen ® Endo F3 cleaves free or asparagine-linked triantennary or α-(1-6)-fucosylated biantennary oligosaccharides, as well as triaminoglycosylated chitobiose core structures. Non-fucosylated biantennary glycans are also cleaved, but at a 40-fold slower rate. 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, PNGaseF removes the oligosaccharide intact. α1-3 fucosylation inhibits the enzyme activity and it is inactive against oligomannose and hybrid molecules.

Specifications:

Rhinogen ^® Endoglycosidase F3 (Endo F3) packaging specifications are as follows:

Stored in 20 mM Tris-HCl, pH 7.5, without preservatives

Supporting reagents:

The reagents provided are as follows:

Product Source :

Endo F3 was recombinantly expressed in E. coli, with a theoretical molecular weight of approximately 74 kDa , an MBP fusion protein at the N-terminus and a 6×His tag at the C-terminus.

Product Quality:

1. High purity: detected by SDS-PAGE, purity ≥90%;

2. High activity: specific activity>4000U/mg, activity>8000U/ml;

3. High stability: Each batch of products undergoes strict quality control to achieve product batch-to-batch stability.

Enzyme activity definition:

The amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 μM porcine fibrinogen in 1 minute at 37˚C, pH 4.5 .

application:

1. Glycomics;

2. Glycoform analysis and determination of glycosylation sites;

3. Proteomics;

4. Mass spectrometry applications.

Storage conditions:

Transported in ice packs, can be stored at 2-8℃ for 12 months. Avoid repeated freezing and thawing.

Precautions for use:

1. For different glycoprotein samples, it is necessary to experiment to find the most suitable enzyme concentration and reaction time.

2. The reaction system can be expanded linearly.

3. To prevent microbial contamination, use sterile methods whenever possible.

4. Higher enzyme concentrations can improve the digestion efficiency of individual glycoproteins and need to be optimized based on actual conditions.

5. Appropriate aliquoting should be performed to reduce the loss of activity caused by multiple freeze-thaw cycles.

6. This product is for research use only and is not intended for human or animal diagnosis or treatment.

Frequently asked questions

Q1: What is a good positive control for Endo F3?

A1: Porcine fibrinogen is a glycoprotein containing fucosylated biantennary N-linked glycans and can be used as a positive control for Endo F3.

Q2: Will detergents inhibit Endo F3 activity?

A2: Endo F3 can only be used under non-denaturing (native) conditions. Detergents, such as SDS, will inhibit the activity of Endo F3.

Q3: What is the optimal pH value for Endo F3 activity?

A3: The optimal pH for Endo F3 to cleave fucosylated biantennary (i.e., porcine fibrinogen) and triantennary glycans (i.e., fetuin) is pH 4.5 (10× Glycobuffer 4). However, fucosylated biantennary glycans can also be cleaved by Endo F3 at pH 6.0 (10× Glycobuffer 3).

Q4: What is the difference between PNGase F and Endo F3?

A4: PNGase F cleaves between the innermost GlcNAc and asparagine residues of high-mannose, hybrid and complex oligosaccharides from N-linked glycoproteins, whereas Endo F3 is highly specific for removing N-linked glycans within the chitobiose core of glycoproteins containing fucosylated bi-antennary and tri-antennary oligosaccharides.