PNGase F, IC-1226
Product Introduction
PNGase F (Peptide-N-Glycosidase F) is a highly specific endoglycosidase isolated from Flavobacterium meningosepticum that cleaves the amide bond between the innermost N-acetylglucosamine (GlcNAc) of the oligosaccharide core and the asparagine residue of glycoproteins. This enzyme releases intact N-linked oligosaccharides from glycoproteins and glycopeptides with broad substrate specificity, recognizing high-mannose, hybrid, and complex-type N-glycans. PNGase F is the most widely used enzyme for complete deglycosylation of N-linked glycoproteins and serves as an essential tool in glycobiology, proteomics, and biopharmaceutical research. Applications include analysis of protein glycosylation patterns, determination of glycosylation site occupancy, structural characterization of released oligosaccharides by mass spectrometry or HPLC, and removal of glycans to facilitate protein crystallization, mass spectrometry analysis, and electrophoretic mobility shift assessment. The enzyme is supplied in an optimized storage buffer and is free of contaminating protease activity, ensuring reliable and specific N-glycan release without protein degradation.
Product Features
1. Broad N-Glycan Specificity: Cleaves all types of N-linked oligosaccharides including high-mannose, hybrid, and complex-type glycans from the asparagine residue.
2. Complete Deglycosylation: Efficiently releases intact N-linked oligosaccharides under native or denaturing conditions, enabling complete removal of glycans for downstream analysis.
3. High Specificity: Does not cleave O-linked glycans or modify the peptide backbone, ensuring protein structural integrity is preserved.
4. Protease-Free: Stringently purified to eliminate contaminating protease activity, preventing protein degradation during prolonged incubations.
5. Essential Glycobiology Tool: Widely used for glycosylation analysis, glycoprotein characterization, and biopharmaceutical quality control.
Specifications
Size: 15,000 units
Source: Flavobacterium meningosepticum
Enzyme Type: Endoglycosidase (EC 3.5.1.52)
Reaction Specificity: Cleaves N-linked glycan at Asn-GlcNAc amide bond
Form: Liquid enzyme solution
Storage and Stability
Storage Conditions: Store at 4°C for short-term use (up to 3 months) or at -20°C for long-term storage. Do not store at -80°C. Avoid repeated freeze-thaw cycles.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed.
Protocol (For Reference Only)
Important: PNGase F activity is affected by buffer composition, incubation temperature, and protein substrate structure. For optimal deglycosylation, glycoproteins should be completely denatured unless native deglycosylation is specifically required.
1. Denaturing Deglycosylation Protocol (Recommended for Most Applications):
a. Glycoprotein Denaturation: Dissolve 1-50 µg of glycoprotein in 1× Denaturing Buffer (e.g., 50 mM sodium phosphate, pH 7.5, 0.1% SDS, 50 mM DTT or β-mercaptoethanol) in a total volume of 10-20 µL. Heat at 95-100°C for 5-10 minutes to completely denature the protein. Allow to cool to room temperature.
b. Detergent Addition: Add 1/10 volume of 10-15% NP-40 or Triton X-100 to the denatured protein mixture to sequester SDS and prevent SDS inhibition of PNGase F activity.
c. Enzyme Addition and Incubation: Add 1-2 µL of PNGase F (1,000-5,000 units) to the reaction mixture. Incubate at 37°C for 1-3 hours. For complex or heavily glycosylated proteins, incubation overnight at 37°C is recommended.
2. Native Deglycosylation Protocol (for Native Protein Analysis):
a. Reaction Setup: Dissolve 1-50 µg of native glycoprotein in 1× Native Reaction Buffer (e.g., 50 mM sodium phosphate, pH 7.5, or 50 mM Tris-HCl, pH 7.5) in a total volume of 10-20 µL.
b. Enzyme Addition and Incubation: Add 2-5 µL of PNGase F (2,000-10,000 units) to the reaction mixture. Incubate at 37°C for 4-24 hours. Note: Native deglycosylation requires longer incubation times and higher enzyme amounts than denaturing conditions.
3. Deglycosylation Monitoring: Assess deglycosylation efficiency by SDS-PAGE followed by Coomassie staining or Western blot. Complete deglycosylation is evidenced by a shift in protein molecular weight corresponding to the removal of N-linked glycans. For glycoproteins with multiple glycosylation sites, multiple intermediate bands may be observed during partial deglycosylation.
4. Downstream Analysis: Following deglycosylation, proceed directly to SDS-PAGE, mass spectrometry, HPLC, or other downstream applications. Released oligosaccharides may be recovered from the reaction mixture by ethanol precipitation or solid-phase extraction for structural analysis.
Precautions
1. SDS concentrations above 0.1% inhibit PNGase F activity. If SDS is used for denaturation, always add NP-40 or Triton X-100 at a detergent-to-SDS ratio of at least 2:1 to sequester SDS before adding the enzyme.
2. Reducing agents (DTT, β-mercaptoethanol) at concentrations above 50 mM may partially inhibit PNGase F. Use the recommended concentration of 50 mM for optimal activity.
3. Do not store the enzyme at -80°C as this may cause loss of activity. For long-term storage, store at -20°C in small aliquots.
4. The enzyme is active over a pH range of 6.0-9.0, with optimal activity at pH 7.5. Avoid buffers with high concentrations of salts, chelating agents, or denaturants.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What types of N-glycans does PNGase F cleave?
A1: PNGase F cleaves all major types of N-linked glycans including high-mannose, hybrid, and complex-type oligosaccharides. It does not cleave N-linked glycans where the core GlcNAc is α(1-3)-fucosylated, which is a common modification in plant and insect glycoproteins but rare in mammalian glycoproteins.
Q2: Why is a detergent needed after SDS denaturation?
A2: SDS at concentrations above 0.1% denatures and inactivates PNGase F. Adding NP-40 or Triton X-100 at a ratio of at least 2:1 (detergent:SDS) forms mixed micelles that sequester SDS, preventing enzyme inhibition while maintaining protein denaturation.
Q3: Can PNGase F deglycosylate native (non-denatured) proteins?
A3: Yes. Native deglycosylation is possible but requires significantly higher enzyme concentrations and longer incubation times (4-24 hours) compared to denaturing conditions. The efficiency of native deglycosylation varies depending on the accessibility of the glycosylation sites on the folded protein.
Q4: How many units of PNGase F should I use per reaction?
A4: For denaturing conditions, 1,000-5,000 units are typically sufficient for 1-50 µg of glycoprotein. For native conditions, 2,000-10,000 units are recommended. One unit is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 nmol of denatured RNase B in 1 minute at 37°C.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of glycoprotein substrates, optimization of deglycosylation conditions is recommended for specific proteins.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-1226
Product Name: PNGase F
Size: 15,000 units
Price: CNY ¥3245.00 / USD $324.50 / EUR �390.00 / JPY ¥58500.00
2023 Version
