Product Class: Other

PNGase F, Recombinant
NEBU cloned at NEB recombinant 37 75 Heat

Product Introduction

PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F, Recombinant is a recombinant amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.

  • Recombinant enzyme; guaranteed endoglycosidase F1-, F2- or F3-free
  • Leaves N-glycan core oligosaccharides intact and suitable for further analysis
  • ≥ 95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Stored in 50% glycerol
  • Optimal activity and stability for up to 24 months
  • Can be used under native or denaturing conditions
Catalog # Size Concentration
P0708S 15000.0 units 500000 units/ml
P0708L 75000.0 units 500000 units/ml

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Product Information

Description

Peptide: N-Glycosidase F, also known as PNGase F, is a recombinant amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1).

Product Source

Cloned from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) and expressed in E. coli (2).

Specificity

This product is related to the following categories:
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Expression Systems,
Glycan Sequencing,
Proteomics,
Recombinant Glycoprotein Expression,
Glycoprotein Analysis

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • P0708S     -20    
      PNGase F, Recombinant P0708SVIAL -20 1 x 0.03 ml 500,000 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
      NP-40 B2704SVIAL -20 1 x 1 ml 10 %
  • P0708L     -20    
      PNGase F, Recombinant P0708LVIAL -20 1 x 0.15 ml 500,000 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
      NP-40 B2704SVIAL -20 1 x 1 ml 10 %

Properties & 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

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2
50 mM Sodium Phosphate
(pH 7.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

75°C for 10 minutes

Molecular Weight

Apparent: 36000 daltons

Application Features

  • Removal of carbohydrate residues from proteins

Product Notes

  1. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  2. Since PNGase F, Recombinant activity is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time.
  3. PNGase F, Recombinant will not cleave N-linked glycans containing core α1-3 Fucose.

References

  1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  2. Chen, M. New England Biolabs, Inc., Unpublished observation

Protocols, Manuals & Usage

Protocols

  1. PNGase F Protocol

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Is there a difference between PNGase F (P0704/P0705) and PNGase F, Recombinant (P0708/P0709)?
  2. I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem?
  3. What happens to the asparagine after PNGase removes the sugar?
  4. Why is protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein. Can a protease inhibitor cocktail be used in a PNGase F, Recombinant reaction?
  5. What is the difference between PNGase F, Endo H and O-Glycosidase?
  6. Does PNGase F work in Urea?
  7. What are the typical reaction conditions for PNGase F, Recombinant?
  8. How much PNGase F, Recombinant should I use to remove my carbohydrate under native or DTT denaturing conditions?
  9. How do I inhibit PNGase F, Recombinant?
  10. Is PNGase F, Recombinant compatible with downstream analysis such as HPLC and Mass Spectrometry?
  11. What are Glycosidases and their uses?
  12. Do detergents inhibit exoglycosidases/endoglycosidases?
  13. 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?
  14. What is a good endoglycosidase substrate?

Tech Tips

  1. You can use this enzyme under native or denaturing conditions
  2. Under native conditions we recommend adding more enzyme and using longer incubation times
  3. 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.
  4. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose (PNGase A must be used in this instance)
  5. A good positive control substrate is RNase B

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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]

Certificate Of Analysis

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

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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