Product Class: Other

Rapid™ PNGase F (non-reducing format)
NEBU cloned at NEB recombinant 50 75 Heat

Product Introduction

Deglycosylation in minutes for intact antibody analysis

Developed for proteomic applications, Rapid PNGase F (non-reducing format) enables complete and rapid deglycosylation, while preserving disulfide bonds. This facilitates high throughput proteomics applications and methods for antibody characterization by mass spectrometry, such as intact mass analysis. Rapid PNGase F (non-reducing format) combines the advantages of Rapid PNGase F (fast processing time), with non-reducing conditions, preserving quaternary structure.

  • Complete deglycosylation of antibodies and immunoglobulin fusion proteins in 15 minutes
  • Recombinant enzyme; guaranteed endoglycosidase F1, F2 or F3 free
  • All reaction components are compatible with HPLC and mass spectrometry analysis
  • ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Optimal activity and stability for up to 12 months
  • Fast reaction setup
Catalog # Size Concentration
P0711S 50.0 reactions

Featured Videos

View Video Library
  • Glycobiology_Animation_thumb

    Overview of Glycobiology

  • PNGase_OneStep_720

    One-step protocol for deglycosylation with Rapid PNGase F

  • PNGase_TwoStep_720

    Two-step protocol for deglycosylation with Rapid PNGase F

Product Information

Description

PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F digestion deaminates the aspargine residue to aspartic acid, and leaves both the protein and the oligosaccharide intact, keeping them suitable for further analysis (1).
Developed for proteomic applications, Rapid PNGase F (non-reducing format) is a reformulated version of Rapid PNGase F (NEB #P0710 ) that allows the complete and rapid deglycosylation of antibodies and fusion proteins in minutes, while preserving disulfide bonds. All N-glycans are released rapidly and without bias, facilitating high throughput proteomics applications and methods for antibody characterization by mass spectrometry such as intact mass analysis. Rapid PNGase F (non-reducing format) combines the advantages of Rapid PNGase F (fast processing time), with the non-reducing conditions preserving quaternary structure.

ESI-TOF analysis of an antibody before and after treatment with Rapid PNGase F (non-reducing format).
ESI-TOF analysis of an antibody before and after treatment with Rapid PNGase F (non-reducing format).

Specificity

Rapid PNGase F (non-reducing format) cleaves all complex, hybrid and high-mannose type glycans from antibodies and related proteins. Core α1,3 fucosylation (found in immunoglobulins expressed in plant or insect cells) is resistant to both Rapid PNGase F and PNGase F.
Rapid PNGase F (non-reducing format)
This product is related to the following categories:
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Expression Systems,
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
  • P0711S     4    
      Rapid™ PNGase F (non-reducing format) P0711SVIAL 4 1 x 0.05 ml 50 reactions
      Rapid PNGase F (non-reducing format) Buffer B0717SVIAL 4 1 x 0.2 ml 5 X

Properties & Usage

Reaction Conditions

1X Rapid PNGase F (non-reducing format) Buffer
Incubate at 50°C

Storage Buffer

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

Heat Inactivation

75°C for 10 minutes

Application Features

Rapid PNGase F (non-reducing format) is effective on a variety of antibodies despite their structural diversity. Intact protein characterization using Rapid PNGase F (non-reducing format) is convenient, and complete (reducing biased results). The fast processing time decreases the risk of artifacts and unwanted protein modifications (oxidation, deamidation) that could be introduced by lengthy incubations with standard N-glycan removal protocols. Samples are ready to be prepared for downstream chromatography or mass spectrometry analysis, providing accurate and reproducible results.

For streamlined proteomic workflows, Rapid PNGase F (non-reducing format) is directly compatible with Trypsin-ultra™, Mass Spectrometry Grade (NEB #P8101 ). 

Product Notes

  1. Storage: It is recommended to store this kit at 4°C. All components will be stable for at least one year if stored correctly. Do not freeze.
  2. The pre-incubation step at 75°C is strictly required. Deglycosylationtakes place once the enzyme is added and incubated at 50°C.
  3. The target protein should be in a solution compatible with Rapid PNGase F(non-reducing format) activity. Avoid buffers containing SDS, as itinhibits PNGase F. Common stabilizing reagents such as Tween, TritonX-100, NP-40, octyl glucoside and non-detergent sulfobetaine, as well astraces of organic solvents, can prevent optimal rapid deglycosylation.
  4. Buffer exchange (by dialysis, spin columns, or microfiltration) isrecommended to prepare samples for liquid chromatography/massspectrometry.

References

  1. Maly, F. et al. (1989). Anal. Biochem. 180, 195-204.

Protocols, Manuals & Usage

Protocols

  1. Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
  2. Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
  3. Glycoproteomics: Buffer Exchange Protocols (P0711)

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the difference between Rapid PNGase F and Rapid PNGase F (non-reducing format)?
  2. What is the difference between Rapid PNGase F (non-reducing format) and Endo S (Endo S, NEB# P0741)?
  3. What are the components of Rapid PNGase F (non-reducing format)?
  4. How much antibody sample can be deglycosylated with Rapid PNGase F (non-reducing format)?
  5. What is a good control substrate for Rapid PNGase F?
  6. What should I do if my target protein is only partially deglycosylated with Rapid PNGase F (non-reducing format)?
  7. What buffers or additives should be avoided when using Rapid PNGase F (non-reducing format)?
  8. How can I test if the Rapid PNGase F (non-reducing format) reaction is complete in 10 minutes?
  9. Is Rapid PNGase F (non-reducing format) compatible with downstream mass spectrometry analysis?
  10. Is Rapid PNGase F (non-reducing format) only suitable for deglycosylation of antibodies?
  11. Are longer incubation times detrimental to the Rapid PNGase F (non-reducing format) reaction?
  12. What is the reaction temperature range for Rapid PNGase F (non-reducing format)?
  13. What happens to the asparagine residue after Rapid PNGase F removes the sugar?
  14. Can a protease inhibitor cocktail be used in a Rapid PNGase F (non-reducing format) reaction?
  15. Is the Rapid PNGase F (non-reducing format) reaction compatible with Trypsin-ultra™ (NEB# P8101)?

Tech Tips

If needed, reaction volumes can be scaled up or down, adjusting the amount of enzyme accordingly. Altering the components ratio will lead to suboptimal results (for instance, using a larger reaction volume with only 1 µl of enzyme).
Reactions can be set up at room temperature.The pre-incubation step at 75°C is strictly required. The deglycosylation step takes place once Rapid PNGase F (non-reducing format) is added and incubated at 50°C.
Typically, the reaction is completed after 10 minutes at 50°C. Incubation longer than 15 minutes will not result in further deglycosylation.
If the reaction is not complete, try increasing the amount of Rapid PNGase F (non-reducing format) Buffer (up to 4 µl in a 10 µl total volume).
For complex substrates, reducing agents are sometimes strictly required. In those cases, complete deglycosylation under non-reducing conditions might not be feasible. Rapid PNGase F (NEB #P0710 ) can be used instead.

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.

Trademarks

NEW ENGLAND BIOLABS® is a registered trademark of New England Biolabs, Inc.
RAPID™ and ULTRA™ are trademarks of New England Biolabs, Inc.

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