PNGase F (Glycerol-free), Recombinant

Catalog # Concentration Size List Price Quantity Your Price
P0709L 500000 units/ml 75000 units $1,072.00
$964.80
P0709S 500000 units/ml 15000 units $267.00
$240.30
Catalog # Size List Price Your Price
P0709L 75000 units $1,072.00
$964.80
P0709S 15000 units $267.00
$240.30
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F (Glycerol-free), 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
  • 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
  • Can be used under native or denaturing conditions
  • Optimized for deglycosylation of glycoproteins; leaves N-glycan core oligosaccharides intact and suitable for further analysis
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Peptide: N-Glycosidase F, also known as PNGase F, is a recombinant 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).

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

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  PNGase F (Glycerol-free), Recombinant P0709SVIAL 4 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 %
  PNGase F (Glycerol-free), Recombinant P0709LVIAL 4 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 %
Application Features
  • Removal of carbohydrate residues from proteins

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.

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
pH 7.5 @ 25°C

Heat Inactivation

75°C for 10 minutes

Molecular Weight

Apparent: 36000 daltons

Unit Assay Conditions

10 μg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F (Glycerol-free), Recombinant are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.

Notes
  • To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  • Since PNGase F (Glycerol-free), 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.
  • PNGase F (Glycerol-free), Recombinant will not cleave N-linked glycans containing core α1-3 Fucose.
  • Recommended storage temperature is 4°C, avoid repeat freeze-thaw cycles.
References
  • Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  • Chen, M. New England Biolabs, Inc., Unpublished observation
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 Control Assay
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]
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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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