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  • Remove-iT® Endo D

    Description

    Remove-iT® Endo D, also known as Endoglycosidase D, is a recombinant glycosidase, which cleaves within the chitobiose core of paucimannose N-linked glycans, with or without extensions in the antennae. Remove-iT Endo D 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

    A truncated Endo D gene cloned from Streptococcus pneumoniae and expressed in E. coli as a fusion to chitin binding domain (2)

    Specificity

    Detailed Specificity

    Figure 1: Detailed specificity of Remove-iT Endo D. The reaction contained 0.08 mU of α1-2 Mannosidase (Prozyme #GKX-5009), 50 units of Remove-iT Endo D and 2 μl 0.5 M Na0Ac pH 5.0 buffer in a total reaction volume of 10 μl. Reactions were incubated at 37°C for 2 hours. Following removal of the bottom branch, Remove-iT Endo D is active on a complex upper arm (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    G7 Reaction Buffer10X
    DTT10X

    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 glycosidase-trimmed (trimannosyl core) Fetuin in 1 hour at 37°C in a total reaction volume of 10 μl.

    Unit Definition Assay
    10 μg of glycosidase-trimmed (trimannosyl core) Fetuin are denatured with 1X DTT at 95°C for 3 minutes. After the addition of 1X G7 Reaction Buffer, two-fold dilutions of Remove-iT Endo D are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

    Reaction Conditions
    1. Combine 10–20 µg of glycoprotein, 1 µl of 10X DTT and H20 (if necessary) to make a 10 µl total reaction volume.
    2. Denature the glycoprotein by heating the reaction at 95°C for 3 minutes.
    3. Make a total reaction volume of 20 µl by adding 2 µl 10X G7 Reaction Buffer, H20 and 1–5 µl Remove-iT Endo D.
    4. Incubate the reaction at 37°C for 1 hour

    Storage Temperature

    4°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 10 min

    Molecular Weight

    Apparent: 140000 daltons

    Quality Control

    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. To deglycosylate a native glycoprotein, longer incubation time, as well as more enzyme, may be required.
    2. Remove-iT Endo D is not recommended for use with Glycoprotein Denaturing Buffer containing both SDS and DTT, as Remove-iT Endo D is inhibited by SDS and, unlike other endoglycosidases, NP-40 does not counteract the SDS inhibition.
    3. Removal of Remove-iT Endo D from the deglycosylation reaction can be scaled up linearly with larger amounts of chitin magnetic beads.
    4. Chitin Magnetic Beads Binding Capacity is 0.4 μg/μl of CBD-tagged protein.
    5. 50 μl of chitin slurry are sufficient to remove 1-5 μl of Remove-iT Endo D (50-250 units)

    References

    1. McLeod, E., Shi, S. and Magnelli, P., New England Biolabs, Inc., unpublished results. Unpublished observation
    2. 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. What is the tag on Remove-iT® Endo D?
    2. What is the difference between PNGase F, Endo S and Endo D?
    3. What is a typical reaction protocol for Remove-iT® Endo D?
    4. Will SDS inhibit Remove-iT® Endo D?
    5. How do I eliminate Remove-iT® Endo D from a reaction?
    6. What is the binding capacity of the Magnetic Chitin Beads used to remove Endo D?
    7. Is Remove-iT® Endo D compatible with downstream analysis such as HPLC and Mass Spectrometry?
    1. Reaction Conditions for Remove-iT® Endo D (P0742)
    2. Remove-iT® Endo D Removal Magnetic Chitin Bead Protocol (P7042)