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Cat. No.: RPNKD-25 (for 25T)

Cat. No.: RPNKD-100 (for 100T)

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

Description

Rapid PNGase F Deglycosylation Kit (Denaturing Method) is an efficient and fast deglycosylation reagent kit. In contrast to traditional PNGase F deglycosylation kits that may take hours or even overnight to remove N-glycosylation modifications, this kit requires only a 10-minute incubation at 50ºC to achieve removal of N-glycan chains from glycoproteins. This significantly shortens the sample processing time, and the treated samples can be used for SDS-PAGE, Western blotting, High-Performance Liquid Chromatography (HPLC), or Mass Spectrometry analysis.

The kit can also be indirectly used for the detection of glycosylation levels in antibodies, immunoglobulin fusion proteins, or other glycoproteins.

Glycosylation is a post-translational modification (PTM) that occurs after protein translation and is present in nearly all living organisms, including eukaryotes, true bacteria (Eubacteria), and archaea (Archaea). The diversity of glycosylation modifications can alter the protein's quality and charge, playing crucial roles in protein classification, immune recognition, receptor binding, inflammation, pathogenicity, and many other biological processes. Recent research indicates that nucleic acids also undergo glycosylation modifications.

Regulating and controlling glycosylation modifications during the production of antibody drugs have significant impacts on the antibody's activity, pharmacokinetics, and immunogenicity. For example, the conserved N-glycan chain at Asn297 in the Fc portion of IgG and additional N-glycan chains in many antibodies may be crucial for the antibody's activity, half-life, and immune response. Variability during cell culture processes may further contribute to the diversity of antibody glycosylation modifications, making the characterization and quality control of N-glycan chains a critical quality attribute (CQA) for antibody drugs.

Rapid PNGase F, similar to PNGase F, is functionally equivalent and expressed through E. coli recombinant expression. It can cleave at the connection sites of N-acetylglucosamine (GlcNAc) and asparagine (Asn) residues at the innermost end of almost all types of N-glycans, including high mannose, hybrid, and complex oligosaccharides. This removes N-linked oligosaccharides, separating protein and glycan modifications. The Rapid PNGase F provided in the kit has a His-tag at the N-terminus, allowing for removal through His antibody agarose gel, magnetic beads, or nickel columns after the reaction.

The in vitro enzymatic deglycosylation reaction can be categorized into denaturing deglycosylation and non-denaturing deglycosylation based on the different treatment methods for glycoproteins or glycopeptides. Denaturing deglycosylation reaction involves the denaturation of proteins, disrupting the tertiary structure and exposing internal sequences, allowing the removal of all glycosylation modifications. Non-denaturing deglycosylation reaction, on the other hand, preserves the protein's native conformation, where exposed glycosylations in the tertiary structure are removed, while those located internally remain intact. The enzymatic activity or antigenicity of the protein is likely unaffected in non-denaturing conditions. Users can choose between the Rapid PNGase F Deglycosylation Kit (Denaturing Method) and the Rapid PNGase F Deglycosylation Kit (Native Method) based on their experimental objectives.

Components

• Rapid PNGase F
• Denaturing Reaction Buffer (5X)

Storage

Store at -20ºC, valid for one year.

Notes

• Rapid PNGase F cannot hydrolyze N-glycosides containing core α1-3 Fucose (common in plant and insect glycoproteins; in such cases, use PNGase A).
• Avoid using buffers containing SDS, as SDS inhibits the enzymatic activity of Rapid PNGase F.
• Tween, Triton X-100, NP-40, Octyl glucoside, non-detergent sulfobetaine, and organic solvents can all inhibit the enzymatic activity of Rapid PNGase F.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food, or drugs. It should not be stored in a regular household.
• For your safety and health, please wear laboratory attire and disposable gloves during operation.

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

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