Endo Hf is a recombinant protein fusion of Endoglycosidase H and maltose binding protein. Endo Hf cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins equally as well as Endo H.
MBP-fusion protein for easy removal
Recombinant enzyme with no detectable exoglycosidase 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 24 months
Product Information
Endo Hf is a recombinant protein fusion of Endoglycosidase H and maltose binding protein. Endo Hf cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins (1) equally as well as Endo H.
60 μg of RNase B was incubated with 3,000 units of Endo H or Endo Hf under standard assay conditions Aliquots were removed at various time points and measured for released carbohydrate. [Dubois et al. (1956) Anal. Chem. 28, 350-356].Mobility Shift Analysis 1 unit of Endo H, Endo Hf or PNGase F was incubated per 10 µg of RNase B under standard assay conditions. At various time points, aliquots were removed and analyzed on a 10-20% SDS-gel for carbohydrate (CHO) release. 1 unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 µg of RNase B in one hour at 37°C.
Product Source
Cloned from Streptomyces picatus (2) and overexpressed in E.coli (3).
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
P0703S
-20
Endo Hf
P0703SVIAL
-20
1 x 0.1 ml
1,000,000 units/ml
GlycoBuffer 3
B1720SVIAL
-20
1 x 1 ml
10 X
Glycoprotein Denaturing Buffer
B1704SVIAL
-20
1 x 1 ml
10 X
P0703L
-20
Endo Hf
P0703LVIAL
-20
1 x 0.5 ml
1,000,000 units/ml
GlycoBuffer 3
B1720SVIAL
-20
1 x 1 ml
10 X
Glycoprotein Denaturing Buffer
B1704SVIAL
-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 denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 µl (10 NEB units = 1 IUB milliunit).
Unit Definition Assay: 10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After the addition of 1X GlycoBuffer 3, two-fold dilutions of Endo Hf are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.
1X Glycoprotein Denaturing Buffer 0.5% SDS 40 mM DTT
To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
Activity at different temperatures (measured after a 1 hour incubation of glycosidase and denatured RNase B at the given temperature): 37°C - 100%; 30°C - 65%; 25°C - 40%; 17°C - 25% and 2°C - 0%.
Typical reaction conditions: Please see the Protocol Tab.
References
Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
Robbins, P. et al. (1984). J. Biol. Chem. 259, 7577-7583.
Guan, C. and Wong, S. New England Biolabs, unpublished Observations. Unpublished observation
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Specifications & Change Notifications
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