PNGase F

unique bufferincubation tempheat inactivation
Item# Description List Price Web Price Qty
P0704L PNGase F (native) - 75,000 units $762.00 $685.80 ADD TO CART
P0704S PNGase F (native) - 15,000 units $191.00 $171.90 ADD TO CART
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
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 that 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. This modification is most commonly found in plant and some insect glycoproteins.

Product Source

PNGase F is purified from Flavobacterium meningosepticum (3) and it is free of proteases and Endo F activities.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C) Concentration
Glycoprotein Denaturing Buffer 10X
NP-40 10%
G7 Reaction Buffer 10X

Advantages and Features

Applications


Glycoprotein analysis

  • Removal of high mannose, hybrid, and complex N-glycans from glycoproteins
  • Free of contaminants (Endo F, proteases, etc.)

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 (65 NEB units = 1 IUB milliunit). 

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 G7 Reaction Buffer, 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 G7 Reaction Buffer
Incubate at 37°C

1X G7 Reaction Buffer:
50 mM sodium phosphate
pH 7.5 @ 25°C

Storage Temperature

-20°C

Storage Conditions

20 mM Tris-HCl
50 mM NaCl
5 mM Na2EDTA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

75°C for 10 min

Molecular Weight

Apparent: 36000 daltons

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.
  • 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.

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. Typical reaction conditions: Please see Protocols

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.

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

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.
  1. Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
  2. What happens to the asparagine after PNGase removes the sugar?
  3. What are Glycosidases and their uses?
  4. 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?
  5. What are the typical reaction conditions for PNGase F?
  6. Do detergents inhibit exoglycosidases/endoglycosidases?
  7. Does PNGase F work in Urea?
  8. What is a good endoglycosidase substrate?
  9. How do I inhibit PNGase F?
  10. How much PNGase F should I use to remove my carbohydrate under native conditions?
  11. I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
  12. What is the difference between PNGase F, Endo H and O-Glycosidase?
  1. PNGase F Protocol (for P0704 and P0705)
PNGase F
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)
Enzyme activity varies at different temperatures: 37°C - 100%; 30°C - 100%; 23°C - 65%; 17°C - 40% and 3°C - 0%
A good positive control substrate is RNase B