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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. 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
    G5 Reaction Buffer10X
    Glycoprotein Denaturing Buffer10X

    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 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.

    1X Glycoprotein Denaturing Buffer
    0.5% SDS
    40 mM DTT

    Reaction Conditions

    1X G5 Reaction Buffer
    Supplement with 50 mM
    Incubate at 37°C

    1X G5 Reaction Buffer:
    50 mM sodium citrate
    pH 5.5 @ 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

    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. 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

    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 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. What is a good endoglycosidase substrate?
    7. Is EndoH/ Endo Hf inhibited by SDS?
    8. Do detergents inhibit exoglycosidases/endoglycosidases?
    9. What are the typical reaction conditions for Endo Hf?
    10. Are Protease Inhibitors acceptable for use in an Endo H/Hf reaction?
    11. What are Glycosidases and their uses?
    1. Endo H/Endo HProtocol

    Usage Guidelines & Tips

    You can use this enzyme under native or denaturing conditions
    Under native conditions we recommend adding more enzyme and using longer incubation times
    A pH of 5.5 is crucial for enzyme activity (supplied G5 reaction buffer is pH 5.5)
    Enzymatic activity is not affected by SDS
    A good positive control substrate is RNase B