PNGase F (Glycerol-free)

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cloned at neb incubation temp heat inactivation
Catalog #SizeConcentrationPriceQtyAdd to Cart
P0705S15,000 units500,000 units/ml$160.00Add to Cart
P0705L75,000 units500,000 units/ml$640.00Add to Cart
  
Categories:
Endoglycosidases,
Proteome Analysis
Applications:
Biosynthesis of Glycans in Eukaryotes,
Glycoprotein Production in Various Expression Systems,
Proteomics

Description


 

Peptide: N-Glycosidase F, also known as PNGase F, is an amidase which is supplied glycerol free for optimal performance in HPLC intensive methods. PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins (1).

Detailed Specificity
PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α1-3 Fucose residue (2). This modification is most commonly found in plant and some insect glycoproteins.
PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α1-3 Fucose residue (2). This modification is most commonly found in plant and some insect glycoproteins.

Product Source

PNGase F is purified from Flavobacterium meningosepticum (3), free of contaminants (Endo F, proteases, etc.).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Glycoprotein Denaturing Buffer-2010X
NP-40-2010%
GlycoBuffer 2-2010X

Advantages and Features

Applications

  • Removal of N-linked glycans from glycoproteins
  • Preferred formulation for HPLC and MS intensive methods of glycoprotein analysis

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 µl.

Unit Definition Assay:
10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. 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.

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

1X NP-40
1% NP-40 in MilliQ-H2O

Storage Temperature

4°C

Storage Conditions

20 mM Tris-HCl
50 mM NaCl
5 mM EDTA
pH 7.5 @ 25°C

Heat Inactivation

75°C for 10 min

Molecular Weight

Apparent: 36000 daltons

Storage Notes

  • Do not freeze

Notes

  1. Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture. Why this non-ionic detergent counteracts the SDS inhibition is unknown at present.
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. 
  3. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose. 
  4. Repeated freeze thaw cycles degrade enzyme activity over time. 
  5. Typical reaction conditions: Please see Protocol tab

References

  1. Maley, F. et al. (1989). Anal. Biochem.. 180, 195-204.
  2. Tretter, V. et al. (1991). Eur. J. Biochem.. 199, 647-652.
  3. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.

FAQs

  1. Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
  2. Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
  3. Do detergents inhibit exoglycosidases/endoglycosidases?
  4. Does PNGase F work in Urea?
  5. How do I inhibit PNGase F?
  6. How much PNGase F should I use to remove my carbohydrate under native conditions?
  7. What is the difference between PNGase F, Endo H and O-Glycosidase?
  8. I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
  9. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
  10. What is a good endoglycosidase substrate?
  11. What are glycosidases and their uses?

Tech Tips

Don’t freeze-thaw this enzyme.  
You can use this enzyme under native or denaturing conditions.  
Under native conditions we recommend adding more enzyme and using longer incubation times.
PNGase F activity is inhibited by SDS, therefore under denaturing conditions it is essential to have NP-40 present in the reaction mixture in a 1:1 ratio.
PNGase F will not cleave N-linked glycans containing core α1-3 Fucose (PNGase A must be used in this instance).

Protocols

  1. PNGase F Protocol

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Feature Articles

Usage Guidelines & Tips

Interactive Tools

Application Notes

Quality Control

Quality Assurance Statement

  • No contaminating exoglycosidase or Endoglycosidase F1, F2 or F3 activity could be detected. No contaminating proteolytic activity could be detected.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Glycosidase Activity (TLC):
    The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
  • Protease Activity (SDS-PAGE):
    The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.