产品信息

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

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

Mobility Shift Analysis

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 H_f Protocol
  2. Protocol for Endo H/H_f 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 H_f?
  3. I tried the Endo H/H_f on my glycoprotein and it failed. What could be the problem?
  4. How much Endo H/Endo H_f should I use?
  5. Is EndoH/ Endo H_f inhibited by SDS?
  6. What are the typical reaction conditions for Endo H?
  7. Are Protease Inhibitors acceptable for use in an Endo H/H_f 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