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Endoglycosidases >
Endo Hf |
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Prices are in US dollars and valid only for US orders.
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- Specific Activity: Endo Hf - 165,000 units/mg
- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer and 10X Denaturation Buffer
Description:
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.
Endo H and Endo Hf cleave only high mannose structures (n = 2-150, x = (Man)1-2, y = H) and hybrid structures (n = 2, x and/or y = AcNeu-Gal-GlcNAc)
60 mg 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.
Source:
Cloned from Streptomyces plicatus (2) and overexpressed in E.coli (3).
Reagents Supplied:
G5 Reaction Buffer (10X)
Glycoprotein Denaturing Buffer (10X)
Enzyme Properties
Molecular Weight:
Apparent: 70 kDa
Specific Activity:
165,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X G5 Reaction Buffer
Incubate at
37°C.
1X G5 Reaction Buffer:
50 mM sodium citrate
pH 5.5 @ 25°C
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 at 100°C for 10 minutes. After the addition of 1X G5 Reaction Buffer, 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.
Concentration:
1,000,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
5 mM EDTA
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Enzymatic activity is not affected by SDS.
- 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 FAQs.
FAQs
- What is the difference between PNGase F and Endo H?
- What is the difference between Endo H and Endo Hf?
- I tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem?
- How much Endo H/Endo Hf should I use?
- What is the pH range of Endo H/Hf?
- What is a good endoglycosidase substrate?
- Is EndoH/ Endo Hf inhibited by SDS?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- What are the typical reaction conditions for Endo Hf?
- What are Glycosidases and their uses?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
No contaminating endoglycosidase, exoglycosidase or proteolytic activity could be detected.
Glycosidase Assays:
5,000 units of Endo Hf were incubated with 0.1 mM of flourescently-labeled oligosaccharides and glycopeptides, in a 10 µl reaction for 20 hours at 37°C. The reaction products were analyzed by TLC for digestion of substrate.
No other glycosidase activities were detected (ND) with the following substrates:
β-N-Acetylglucosaminidase:
GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND
α-Fucosidase:
Fucα1-2Galβ1-4Glc-AMC ND
Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND
β-Galactosidase:
Galβ1-3GlcNAcβ1-4Galβ1-4Glc-AMC ND
α-Galactosidase:
Galα1-3Galβ1-4GlcNAc-AMC ND
α-Neuraminidase:
Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ 1-4Glc-AMC ND
α-Mannosidase:
Manα1-3Manβ1-4GlcNAc-AMC ND
Manα1-6Manα1-6(Manα1-3)Man-AMC ND
β-Glucosidase:
Glcβ1-4Glcβ1-4Glc-AMC ND
β-Xylosidase:
Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND
β-Mannosidase:
Manβ1-4Manβ1-4Man-AMC ND
Endo F2, F3:
Dansylated fibrinogen biantennary ND
PNGase F:
Fluoresceinated fetuin triantennary ND
Protease Assay:
After incubation of 5,000 units of Endo Hf with 0.2 nmol of a standardized mixture of proteins, for 20 hours at 37°C, no proteolytic activity could be detected by SDS-PAGE.
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.
Companion Products
Endo H
Endoglycosidase Reaction Buffer Pack
PNGase F
PNGase F (Glycerol Free)
RNase B
Endo H crystal (Dr. Patrick Van Roey)
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