Endo H |NEB酶试剂 New England Biolabs

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产品信息

Endo H |
 
Endoglycosidase H is a recombinant glycosidase which cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins.

Endo H | Endo H |

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

Endo H |
Aliquots were removed at various time points and measured for released carbohydrate. [Dubois et al. (1956) Anal. Chem. 28, 350-356].
Mobility Shift Analysis
Endo H |
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-PAGE 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.

产品来源

Cloned from Streptomyces picatus (2) and overexpressed in E.coli (3).

产品类别:
Endoglycosidases Products,
Proteome Analysis Products

应用:
Expression Systems,
Glycan Sequencing,
Proteomics,

Recombinant Glycoprotein Expression,

Glycoprotein Analysis

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P0702S     -20    
        Endo H P0702SVIAL -20 1 x 0.02 ml 500,000 units/ml
        GlycoBuffer 3 B1720SVIAL -20 1 x 1 ml 10 X
        Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
    • P0702L     -20    
        Endo H P0702LVIAL -20 1 x 0.1 ml 500,000 units/ml
        GlycoBuffer 3 B1720SVIAL -20 1 x 1 ml 10 X
        Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X

  • 特性和用法

    单位定义

    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 GlycoBuffer 3, two-fold dilutions of Endo H are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

    1X Glycoprotein Denaturing Buffer
    0.5% SDS
    40 mM DTT

    反应条件

    1X GlycoBuffer 3
    Incubate at 37°C

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

    贮存溶液

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

    热失活

    75°C for 10 minutes

    分子量

    实际: 29 kDa

  • 优势和特性

    应用特性

    • Removal of high mannose N-glycans from glycoproteins

  • 相关产品

    相关产品

    • p0703-endo-hf
    • p0704-pngase-f
    • p0705-pngase-f-glycerol-free
    • RNase B（对照底物）
    • p0706-remove-it-pngase-f
    • Endo S
    • 糖苷内切酶 D

  • 注意事项

    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 FAQs.

  • 参考文献

    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.

操作说明、说明书 & 用法

  • 操作说明

    1. Endo H/Endo Hf Protocol
    2. Protocol for Endo H/Hf Non-Denaturing P0702 and P0703

工具 & 资源

  • 选择指南

    • Endoglycosidase Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between PNGase F, Endo H and O-Glycosidase?
    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. Is EndoH/ Endo Hf inhibited by SDS?
    6. What are the typical reaction conditions for Endo H?
    7. Are Protease Inhibitors acceptable for use in an Endo H/Hf reaction?
    8. What are Glycosidases and their uses?
    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?

  • 实验技巧

    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