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  • Remove-iT® Endo S

    Description

    Remove-iT® Endo S is an endoglycosidase with a uniquely high specificity for removing N-linked glycans from the chitobiose core of the heavy chain of native IgG (1). Remove-iT Endo S 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 Endo S is cloned from Streptococcus pyogenes (1) and overexpressed as a fusion to the chitin binding domain in E. coli (3).

    Specificity

    P0741
    Detailed Specificity
    X = protein, peptide, Asparagine, or free glycan, as Remove-iT Endo S does not have a strict peptide requirement for activity. Remove-iT Endo S is active on a substrate with or without core α(1-6)fucosylation as well as a with or without a bisecting N-acetylglucosamine. Triantennary and tetrantennary sialyated or asialo glycans are not a substrate for Remove-iT Endo S (2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    G6 Reaction Buffer10X

    Properties and Usage

    Unit Definition

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

    Unit Definition Assay
    5 µg of IgG in 1X G6 Reaction Buffer are incubated with two-fold dilutions of Remove-iT Endo S for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

    Reaction Conditions
    1. Combine 100 μg of native IgG, 1 μl of 10X G6 Reaction Buffer and H20 (if necessary) to make a 10 μl total reaction volume.
    2. Add 1 μl Remove-iT Endo S.
    3. Incubate reaction at 37°C for 1 hour.

    Storage Temperature

    4°C

    Storage Conditions

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

    Heat Inactivation

    55°C for 10 min

    Molecular Weight

    Apparent: 136000 daltons

    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.
    • 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. Recommended storage temperature is 4°C, avoid repeat freeze-thaw cycles.
    2. Removal of Remove-iT Endo S from the deglycosylation reaction can be scaled up linearly with larger volumes of chitin magnetic beads.
    3. Chitin Magnetic Beads Binding Capacity is 0.4 µg/µl of CBD-tagged protein.

    References

    1. Collin, M. and Olsén, A. (2001). The EMBO Journal. 20, 3046-3055.
    2. Bielik, A., McLeod, E. and Magnelli, P., New England Biolabs,Inc., unpublished results.. Unpublished observation
    3. McLeod, E., New England Biolabs, Inc., unpublished results.. Unpublished observation

    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 Remove-iT® Endo S tagged?
    2. What is the difference between PNGase F and Endo S?
    3. How much Remove-iT® Endo S should I use to deglycosylate a glycoprotein under native conditions?
    4. What is the preferred substrate for Remove-iT® Endo S?
    5. What are the typical reaction conditions for Remove-iT® Endo S?
    6. How do I eliminate Remove-iT® Endo S from a reaction?
    7. What is the binding capacity of the Magnetic Chitin Beads used to remove Endo S?
    8. Is Remove-iT® Endo S compatible with downstream analysis such as HPLC and Mass Spectrometry?
    9. What are Glycosidases and their uses?
    1. Reaction Conditions for Remove-iT® Endo S (P0741)
    2. Remove-iT® Endo S Removal Magnetic Chitin Bead Protocol (P0741)

    Application Notes