What is PNGase F used for?

PNGase F is an endoglycosidase that specifically removes N-linked glycans from glycoproteins. It is used extensively in workflows for characterizing N-linked glycan structures on therapeutic proteins and for identifying N-linked glycosylation sites in proteomic studies.

Why glycosylation is important in antibody?

Antibody glycosylation defines the functional potential of the antibody by delineating the structure of the antibody Fc region and determining which Fc receptors it can bind to in order to recruit effector cells.

Are antibodies glycosylated?

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy.

How do I get rid of PNGase F?

Reaction Conditions Denature glycoprotein by heating reaction at 55°C for 10 minutes. Make a total reaction volume of 20 μl by adding 2 μl 10X GlycoBuffer 2, H20 and 1–5 μl Remove-iT PNGase F.

Where does PNGase F cleave?

PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.

Is trastuzumab a glycosylation?

Due to its relevance, the ErbB2-targeted monoclonal antibody Trastuzumab (Herceptin) used as breast cancer therapy is one of the main targets in the production of biosimilars. A major challenge is to produce antibodies with the same or the closest N-glycosylation pattern seen in the commercialized drug.

What is the function of PNGase F?

PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides. ≥ 95% purity, as determined by SDS-PAGE and intact ESI-MS.

Does PNGase F cleave glycans containing core α1-3 fucose?

PNGase F will not cleave N -linked glycans containing core α1-3 Fucose. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.

Can PNGase F deglycosylate a native glycoprotein?

To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. PNGase F will not cleave N -linked glycans containing core α1-3 Fucose. Maley, F. et al. (1989).

How to prepare PNGase F with NP-40 and glycobuffer 2?

After the addition of NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture.