Endo F2

Description

Endo F2 is a highly specific recombinant endoglycosidase which cleaves within the chitobiose core of asparagine-linked complex biantennary and high mannose oligosaccharides from glycoproteins and glycopeptides. Endo F2 cleaves biantennary glycans at a rate approximately 20 times greater than high mannose glycans.  The activity of Endo F2 is identical on biantennary structures with and without core fucosylation.  However, Endo F2 is not active on hybrid or tri- and tetra-antennary oligosaccharides. Endo F2 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.

Specificity:
Endo F2 Specificity

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
GlycoBuffer 4-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave > 95% of the carbohydrate from 10 µg Porcine Fibrinogen in 1 hour at 37°C in a total reaction volume of 10 µl.

Reaction Conditions

1X GlycoBuffer 4

1X GlycoBuffer 4:
50 mM sodium acetate
pH 4.5 @ 25°C

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

65°C for 10 min

Molecular Weight

Apparent: 39.8 kDa

Unit Assay Conditions

Two fold dilutions of Endo F2 are incubated with 10µg Porcine Fibrinogen and 1X GlycoBuffer 4 in a 10 µl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via SDS-PAGE.

Notes

  1. The enzyme can be used under denaturing and non-denaturing (native) conditions. However, optimal activity occurs under non-denaturing conditions.
  2. Reactions may be scaled-up linearly to accommodate larger reaction volumes.

References

  1. Guthrie, E. Unpublished results. New England Biolabs, Inc..

FAQs

  1. Is Endo F2 tagged?
  2. What is the difference between PNGase F and Endo F2?
  3. What is the difference between Endo F2 and Endo F3?
  4. How much Endo F2 should I use to deglycosylate a glycoprotein under native conditions?
  5. What is the preferred substrate for Endo F2?
  6. Do detergents inhibit Endo F2 activity?
  7. What is the optimal pH for Endo F2 activity?
  8. How do I eliminate Endo F2 from a reaction?
  9. What is the binding capacity of the Magnetic Chitin Beads used to remove Endo F2?
  10. Is Endo F2 compatible with downstream analysis such as HPLC and Mass Spectrometry?
  11. What are Glycosidases and their uses?

Tech Tips

  • Endo F2 is tagged with a chitin binding domain (CBD) for easy removal from a reaction using chitin magnetic beads (NEB #E8036 )
  • Endo F2 is typically used under non-denaturing (native) conditions, as denaturation is not necessary for optimal activity of the enzyme.
  • Concentrations of 0.5% SDS do not inhibit Endo F2 activity when used in the presence of 1% NP-40.
  • The optimal pH for cleavage of biantennary glycans is pH 4.5 (10X Glycobuffer 4)
  • Although not as optimal, cleavage can also occur at pH 6.0 (10X Glycobuffer 3).

Protocols

  1. Endo F2 Reaction Protocol (P0772)
  2. Removal of Endo F2 by Magnetic Beads (P0772)

Quality Control

Quality Assurance Statement

  • No contaminating endoglycosidase, exoglycosidase, or 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. Further information regarding NEB product quality can be found here.
  • 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.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

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]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.