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Cat. No.: PNKMB-25 (for 25T)

Cat. No.: PNKMB-100 (for 100T)

Cat. No.: PNKMB-500 (for 500T)

Description

PNGase F Deglycosylation Kit with Magnetic Beads, also known as the PNGase F Glycan Cleavage Kit with Magnetic Beads or PNGase F Releasing Kit with Magnetic Beads, is a deglycosylation reagent kit designed to cleave N-glycosidic bonds from glycoproteins or glycopeptides under both native or denaturing conditions, effectively removing N-linked glycan chains. The PNGase F in this kit is biotinylated (Biotinylated PNGase F) and can bind to streptavidin magnetic beads. Subsequently, Biotinylated PNGase F can be rapidly and conveniently removed from the reaction system using magnetic separation devices such as a magnetic rack, eliminating the need for traditional column purification. This greatly simplifies the sample processing steps.

PNGase F, also known as Peptide N-Glycosidase F or N-acylamide hydrolase F, is an enzyme derived from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) via recombinant expression in Escherichia coli. PNGase F can cleave the N-acetylglucosamine (GlcNAc) and asparagine (Asn) bond at the innermost site of various types of N-linked glycans, including high-mannose, hybrid, and complex oligosaccharides, thus removing N-linked oligosaccharides and separating the protein from its glycan modifications. This enzyme can be used in in vitro reactions to remove N-glycosylation modifications from antibodies, immunoglobulin fusion proteins, or other glycoproteins.

Biotin and streptavidin form a naturally occurring biomolecular interaction system, with an affinity constant (Kd) of 10⁻¹⁵ M, which is one of the strongest known protein-ligand interactions. Streptavidin is a tetrameric protein that can specifically bind up to four biotin molecules with high affinity.

Glycosylation is a type of post-translational modification (PTM) that occurs in nearly all living organisms, including eukaryotes, eubacteria, and archaea. The diversity of glycosylation modifications alters the mass and charge of proteins, playing key roles in protein classification, immune recognition, receptor binding, inflammation, pathogenicity, and many other biological processes [2-4]. Recent studies have also shown that nucleic acids can undergo glycosylation modifications.

The optimal enzyme activity temperature for Biotinylated PNGase F is 37°C, and it exhibits high enzyme activity over a wide pH range (6.0–10.0), temperature range (4–50°C), and ionic strength range (0–1M NaCl).

This kit is designed for the removal of N-linked oligosaccharides (N-glycans) from glycoproteins or glycopeptides. For a reaction using 1 μL of Biotinylated PNGase F (500 U/μL) per 100 μg of glycoprotein at 37°C for 1 hour, the small, medium, and large kit sizes can be used for N-glycan removal from 2.5 mg, 5 mg, and 25 mg of glycoprotein, respectively, which is equivalent to approximately 25, 100, and 500 reactions.

Components

• Biotinylated PNGase F (500U/μl)
• Reaction Buffer (10X)
• Denaturing Buffer (10X)
• Renaturing Buffer (10X)
• Streptavidin Magnetic Beads

Storage

Store at -20ºC, with a one-year shelf life. To avoid repeated freeze-thaw cycles of Biotinylated PNGase F, it is recommended to aliquot the reagent upon first use.

Notes

• Biotinylated PNGase F cannot hydrolyze N-linked glycans containing core α1-3 fucose (commonly found in plant and insect glycoproteins). In such cases, PNGase A should be used instead.
• Before use, streptavidin magnetic beads should be properly resuspended by gently inverting the vial several times to ensure uniform mixing. The mixing process should be done gently, avoiding vigorous vortexing or shaking.
• This product is intended for scientific research use by professionals only. It should not be used for clinical diagnosis or treatment, nor for food or drug applications, and must not be stored in ordinary residential settings.
• For your safety and health, please wear laboratory clothing and disposable gloves during operation.

Related: 

• PNGase F
• PNGase F (with His-tag)
• PNGase F (with His-tag, Glycerol Free)
• PNGase F Deglycosylation Kit with Magnetic Beads
• Rapid PNGase F Deglycosylation Kit (Denaturing Method)
• Rapid PNGase F Deglycosylation Kit (Native Method)

Only for research and not intended for treatment of humans or animals

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