PNGase F Deglycosylation Kit KP2318M

The PNGase F Deglycosylation Kit, also known as PNGase F Glycan Cleavage Kit or PNGase F Releasing Kit, provides a method to cleave N-glycosidic bonds and remove N-glycan chains from glycoProteins or glycopeptides under native or denaturing conditions.

The Peptide N-Glycosidase F (PNGase F) is a recombinant Elizabethkingia miricola glycosyl peptidase expressed and purified from E. coli. PNGase F can cleave at the linkage between N-acetylglucosamine (GlcNAc) and asparagine (Asn) residues of almost all types of N-polysaccharides including high-mannose, hybrid and complex oligosaccharides, to remove N-linked oligosaccharides and separating protein and glycan modifications (Figure 1) [1]. It can be used for in vitro removal of N-glycosylation modifications from Antibodies, immunoglobulin fusion Proteins, or other glycoProteins. Additionally, after the isolation of cells or membrane Proteins with Concanavalin A magnetic beads, PNGase F can be used to dissociate glycoProteins from  Concanavalin A magnetic beads by cleaving N-glycans, thus facilitating subsequent mass spectrometry analysis of membrane proteins [2]. This PNGase F has a N-terminal His tag and can be removed using the a nickel column or magnetic beads/agrose coupled with His Antibodies.

Figure 1. Schematic diagram of N-linked oligosaccharide removal by using koolbio PNGase F Deglycosylation Kit (P2318). The sugar chain can be extended at R1, R2 and R3 positions of pentasaccharide. The R1 position must be vacant or occupied by α-6 fucose and cannot be fanyα-3 fucose. Otherwise, it cannot be cleaved by PNGase F. The R2 and R3 locations can be any monosaccharides or polysaccharides.

Glycosylation is a ubiquitous post-translational modification (PTM) in almost all organisms including eukaryotes, eubacteria, and Archaea. The diversity of glycosylation modifications alters the mass and charge of proteins, playing key biological functions in protein classification, immune recognition, receptor binding, inflammation, pathogenicity, and many other processes [3-5]. Recent studies have shown that nucleic acids can also undergo glycosylation modifications.

The basic information of PNGase F in this kit is shown as below:

PNGase F has an optimal working temperature of 37℃, high enzyme activity in a wide pH range (6.0-10.0), a wide temperature range (4-50℃), and a wide range of ionic strengths (0-1M NaCl).

PNGase F Storage Buffer: 10mM Tris (pH 7.4), 50mM NaCl, 5mM EDTA.

Reaction Buffer (10X): 500mM Sodium Phosphate (pH7.5).

When using 1μl of PNGase F (500U/μl) per 100μg of glycoprotein at 37℃, P2318S, P2318M, and P2318L are sufficient for 25, 100 and 500 deglycosylation reactions, respectively.

Packing List：

Storage Conditions：

Store at -20℃ for up to 1 year.

Precautions：

PNGase F cannot hydrolyze N-glycans containing core α1-3 fucose which is commonly found in plant and insect glycoproteins. To hydrolyze such N-glycans, PNGase A should be used.

For Ultrapure Water, we recommend using the BeyoPure™ Ultrapure Water (DNase/RNase-Free, Sterile)

This product is for R&D only. Not for drug, household, or other uses.

For your safety and health, please wear a lab coat and disposable gloves during the operation.