Endo D is an endoglycosidase that cleaves within the chitobiose core of paucimannose N-linked glycans, with or without extensions in the antennae, from glycoproteins and glycopeptides.
Tagged with a chitin binding domain (CBD) for easy removal
Recombinant enzyme with no detectable exooglycosidase or other endoglycosidase contaminating activities
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
Product Information
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. 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
Specificity
Detailed SpecificityFigure 1: Detailed specificity of Endo D The reaction contained 0.08 mU of α1-2 Mannosidase (Prozyme #GKX-5009), 50 units of 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, Endo D is active on a complex upper arm (1).
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
P0742S
4
Endo D
P0742SVIAL
4
1 x 0.03 ml
50,000 units/ml
DTT
B0706SVIAL
-20
1 x 1 ml
0.4 M
GlycoBuffer 2
B3704SVIAL
-20
1 x 1 ml
10 X
P0742L
4
Endo D
P0742LVIAL
4
1 x 0.15 ml
50,000 units/ml
DTT
B0706SVIAL
-20
1 x 1 ml
0.4 M
GlycoBuffer 2
B3704SVIAL
-20
1 x 1 ml
10 X
Properties & 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.
10 μg of glycosidase-trimmed (trimannosyl core) Fetuin are denatured with 1X DTT at 95°C for 3 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of 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.
To deglycosylate a native glycoprotein, longerincubation time, as well as more enzyme, maybe required.
Endo D is not recommended foruse with Glycoprotein Denaturing Buffercontaining both SDS and DTT, as Endo D is inhibited by SDS and, unlike otherendoglycosidases, NP-40 does not counteractthe SDS inhibition.
Removal of Endo D from thedeglycosylation reaction can be scaled uplinearly with larger amounts of chitin magneticbeads.
Chitin Magnetic Beads Binding Capacity is0.4 μg/μl of CBD-tagged protein.
50 μl of chitin slurry are sufficient to remove 1-5 μl of Endo D (50-250 units)
References
McLeod, E., Shi, S. and Magnelli, P., New England Biolabs, Inc., unpublished results. Unpublished observation
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Specifications & Change Notifications
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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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