Product Class: Other

PNGase A
NEBU cloned at NEB recombinant 37 65 Heat

Product Introduction

PNGase A cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and short complex oligosaccharides such as those found in plant and insect cells from N-linked glycoproteins and glycopeptides. PNGase A differs from PNGase F in that it cleaves N-linked glycans with or without α(1,3)-linked core fucose residues.

  • Cleaves N-glycans from plant and insect derived glycoproteins and glycopeptides
  • Activity is not inhibited by an α1-3 Fucose residue on the chitobiose core
  • Recombinant enzyme with no detectable exoglycosidase or other endoglycosidase contaminating activities
  • Glycerol-free for optimal performance in HPLC and mass spectrometry analysis
  • ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Optimal activity and stability for up to 24 months 
Catalog # Size Concentration
P0707S 150.0 units 5000 units/ml
P0707L 750.0 units 5000 units/ml

Product Information

Description

PNGase A is a recombinant amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and short complex oligosaccharides such as those found in plant and insect cells from N-linked glycoproteins and glycopeptides. PNGase A differs from PNGase F in that it cleaves N-linked glycans with or without α(1,3)-linked core fucose residues.

Substrate Specificity
PNGase A hydrolyzes N-glycan chains from glycoproteins/peptides regardless of the presence of xylose or fucose. [x = H or Man or GlcNAc].

Product Source

Cloned from Oryza sativa (rice) and expressed in Pichia pastoris.
This product is related to the following categories:
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Glycomics and glycoproteomics,
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
  • P0707S     4    
      PNGase A P0707SVIAL 4 1 x 0.03 ml 5,000 units/ml
      GlycoBuffer 3 B1720SVIAL -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 %
  • P0707L     4    
      PNGase A P0707LVIAL 4 1 x 0.15 ml 5,000 units/ml
      GlycoBuffer 3 B1720SVIAL -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 1 µg of denatured recombinant Avidin produced in Maize in 1 hour at 37°C in a total reaction volume of 10 µl.

Reaction Conditions

1X GlycoBuffer 3
Incubate at 37°C

1X GlycoBuffer 3
50 mM sodium acetate
(pH 6 @ 25°C)

Storage Buffer

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

Heat Inactivation

65°C for 10 minutes

Molecular Weight

Apparent: 63.8 kDa

Unit Assay Conditions

1 μg of recombinant Avidin is denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 3, two-fold dilutions of PNGase A are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.

Product Notes

  1. PNGase A is active on both glycoproteins and glycopeptides.
  2. PNGase A cannot cleave larger N-glycans such as those from Fetuin, Fibrinogen, IgG, Lactoferrin and Transferrin.
  3. PNGase A is able to cleave high mannose N-glycan structures from Man 3 up to Man 9.
  4. Reactions may be scaled-up linearly to accommodate larger reaction volumes.

Protocols, Manuals & Usage

Protocols

  1. Reaction Conditions for PNGase A (P0707)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the difference between PNGase F and PNGase A?
  2. Can PNGase A be used under non-denaturing (native) conditions?
  3. Which high mannose structures can PNGase A cleave?
  4. Can PNGase A cleave large, complex oligosaccharides?
  5. What happens to the asparagine after PNGase A removes the sugar?
  6. What is a good PNGase A substrate?
  7. Is PNGase A compatible with downstream analysis such as HPLC and Mass Spectrometry?
  8. How much PNGase A should I use to remove my carbohydrate under native or DTT denaturing conditions?
  9. Why is my 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 A reaction?
  10. What are Glycosidases and their uses?

Tech Tips

NEB's version of PNGase A can cleave glycoproteins, there is no need for tryptic digest prior to deglycosylation.

PNGase A can cleave N-linked glycans containing core α1-3 Fucose.

PNGase A 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.

A good positive control substrate is recombinant avidin from maize or HRP.

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.

Other Products You May Be Interested In