Product Class: Other

Remove-iT® PNGase F
NEBU cloned at NEB 37 75 Heat

Product Introduction

Remove-iT® PNGase F is a tagged version of PNGase F, optimized for easy removal from a reaction. Remove-iT PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.

  • Chitin Binding Domain (CBD) tag ensures easy removal from a reaction
  • 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 12 months
  • Optimized for deglycosylation of glycoproteins; leaves N-glycan core oligosaccharides intact and suitable for further analysis
Catalog # Size Concentration
P0706S 6750.0 units 225000 units/ml
P0706L 33750.0 units 225000 units/ml

Product Information

Description


 
Remove-iT® PNGase F is an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1). Remove-iT PNGase F is tagged with a chitin binding domain (CBD) for easy removal from a reaction and is supplied glycerol free for optimal performance in HPLC and MS intensive methods.

Product Source

Remove-iT PNGase F is purified from Elizabethkingia miricola (formerly Flavobacterium meningosepticum).

Specificity

P0706
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
  • P0706S     4    
      Remove-iT® PNGase F P0706SVIAL 4 1 x 0.03 ml 225,000 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      DTT B0706SVIAL -20 1 x 1 ml 0.4 M
  • P0706L     4    
      Remove-iT® PNGase F P0706LVIAL 4 1 x 0.15 ml 225,000 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      DTT B0706SVIAL -20 1 x 1 ml 0.4 M

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 5 μg of DTT denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl.

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

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

Molecular Weight

Apparent: 41000 daltons

Unit Assay Conditions

5 µg of RNase B are denatured with 1X DTT at 55°C for 10 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of Remove-iT PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

Product Notes

  1. To deglycosylate a native glycoprotein, longerincubation time, as well as more enzyme, maybe required.
  2. Using typical RNase B denaturing conditions with NEB Glycoprotein Denaturing Buffer,containing SDS and DTT, Remove-iT PNGase F yields a higher concentration of 500,000 U/ml.
  3. If using Remove-iT PNGase F under typical PNGase F denaturing conditions, it is essential to have NP-40 in the reaction mixture as Remove-iT PNGase F is inhibited by SDS. It is not known why this non-ionic detergent counteracts the SDS inhibition.
  4. Remove-iT PNGase F will not cleave N-linkedglycans containing core α1-3 Fucose.
  5. Recommended storage temperature is 4°C,avoid repeat freeze-thaw cycles
  6. Removal of Remove-iT PNGase F from the deglycosylation reaction can be scaled up linearly with larger volumes of chitin magnetic beads.
  7. Chitin Magnetic Beads Binding Capacity is0.4 µg/µl of CBD-tagged protein.
  8. To remove 1-5 µl of Remove-iT PNGase F use 50 µl of chitin magnetic beads.

References

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

Protocols, Manuals & Usage

Protocols

  1. Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
  2. Reaction Conditions for Remove-iT® PNGase F (P0706)

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the tag on PNGase F?
  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 is the difference between PNGase F and Remove-iT® PNGase F?
  4. What is the difference between Remove-iT® PNGase F and Endo H?
  5. How much Remove-iT® PNGase F should I use to remove my carbohydrate under native or DTT denaturing conditions?
  6. Does Remove-iT® PNGase F work in Urea?
  7. What are the typical reaction conditions for Remove-iT® PNGase F?
  8. How do I eliminate Remove-iT® PNGase F from a reaction?
  9. What is the binding capacity of the Magnetic Chitin Beads used to eliminate Remove-iT® PNGase F?
  10. Is Remove-iT® PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
  11. 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?
  12. What is a good endoglycosidase substrate?
  13. Do detergents inhibit exoglycosidases/endoglycosidases?
  14. What are Glycosidases and their uses?

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.
REMOVE-IT™ is a trademark of New England Biolabs, Inc.

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