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  • Endo Hf

    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.


    60 μg of RNase B was incubated with 3,000 units of Endo H or Endo Hf under standard assay conditions
    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).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Glycoprotein Denaturing Buffer-2010X
    Glyco Buffer 3-2010X

    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 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

    Reaction Conditions

    1X GlycoBuffer 3 
    Supplement with 50 mM
    Incubate at 37°C

    1X GlycoBuffer 3:
    50 mM sodium acetate
    pH 6 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Apparent: 70 kDa

    Notes

    1. Enzymatic activity is not affected by SDS.
    2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
    3. 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%.
    4. Typical reaction conditions: Please see the Protocol Tab.

    References

    1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
    2. Robbins, P. et al. (1984). J. Biol. Chem. 259, 7577-7583.
    3. Guan, C. and Wong, S. New England Biolabs, unpublished Observations. Unpublished observation

    FAQs

    1. What is the difference between PNGase F and Endo H?
    2. What is the difference between Endo H and Endo Hf?
    3. I tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem?
    4. How much Endo H/Endo Hf should I use?
    5. What is the pH range of Endo H/Hf?
    6. Is EndoH/ Endo Hf inhibited by SDS?
    7. What are the typical reaction conditions for Endo Hf?
    8. Are Protease Inhibitors acceptable for use in an Endo H/Hf reaction?
    9. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
    10. What is a good endoglycosidase substrate?
    11. Do detergents inhibit exoglycosidases/endoglycosidases?
    12. What are glycosidases and their uses?

    Tech Tips

    You can use this enzyme under native or denaturing conditions
    Under native conditions we recommend adding more enzyme and using longer incubation times
    Enzymatic activity is not affected by SDS
    A good positive control substrate is RNase B

    Protocols

    1. Endo H/Endo HProtocol

    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.

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    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.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    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.