产品信息

• 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
  P0733S     -20       O-Glycosidase P0733SVIAL -20 1 x 0.05 ml 4.0E7 units/ml   GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X   Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X   NP-40 B2704SVIAL -20 1 x 1 ml 10 %
  P0733L     -20       O-Glycosidase P0733LVIAL -20 1 x 0.25 ml 4.0E7 units/ml   GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X   Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X   NP-40 B2704SVIAL -20 1 x 1 ml 10 %

• 特性和用法

  单位定义

  One unit is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of neuraminidase digested, non-denatured fetuin (2) in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

  1X Glycoprotein Denaturing Buffer
  0.5% SDS
  40 mM DTT

  1X NP-40
  1% NP-40 in MilliQ-H₂O

  反应条件

  1X GlycoBuffer 2
  Incubate at 37°C

  1X GlycoBuffer 2
  50 mM Sodium Phosphate
  (pH 7.5 @ 25°C)

  贮存溶液

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

  热失活

  65°C for 10 minutes

  分子量

  实际: 147000 daltons

  单位活性检测条件

  Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction mix is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by the Morgan and Elson Assay (1).

• 优势和特性

  应用特性

  • Removal of Core 1 and Core 3 O-linked disaccharide glycans from glycoproteins

• 相关产品

  相关产品

  • O-糖苷酶 & 神经氨酸苷酶套装
  • α2-3,6,8 神经氨酸苷酶
  • p0704-pngase-f
  • p0705-pngase-f-glycerol-free
  • p0703-endo-hf
  • p0702-endo-h

• 注意事项

  1. Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  3. It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (#P0720) or the O-Glycosidase and Neuraminidase bundle (#E0540S)  is recommended.
  4. Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

• 参考文献

  1. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

操作说明、说明书 & 用法

• 操作说明

  1. O-Glycosidase Application Note 1 (P0733)
  2. O-Glycosidase (P0733)
  3. Endo-α-N-Acetylgalactosaminidase Application Note 1

• 应用实例

  • AppNote_Enzymatic_Deglycosylation_of_Protein_Containing_Core_1_O-glycans_with_O-Glycosidase
  • Analysis of a Fusion Protein using the Protein Deglycosylation Mix II and Mass Spectrometry
  • Enzymatic Deglycosylation of a Protein Containing Core 1 O-glycans with O-Glycosidase

工具 & 资源

• 选择指南

  • Endoglycosidase Selection Chart

FAQs & 问题解决指南

• FAQs

  1. What are the typical reaction conditions for O-Glycosidase?
  2. I tried using O-Glycosidase on my glycoprotein and didn’t see removal of the carbohydrate. What could be the problem?
  3. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
  4. Do detergents inhibit O-Glycosidase?
  5. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
  6. Can I double digest PNGase F and O-Glycosidase?
  7. What is the difference between PNGase F, Endo H and O-Glycosidase?
  8. 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?
  9. What are Glycosidases and their uses?
  10. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
  11. How do I inhibit O-Glycosidase?

• 实验技巧

  Don’t forget to include Neuraminidase (P0720) in your reaction! It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides.

  NEB’s O-Glycosidase is the only available O-Glycosidase that catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins

  You can use this enzyme under native or denaturing conditions

  Under native conditions we recommend adding more enzyme and using longer incubation times

  Under denaturing conditions the enzyme activity is increased two-fold. However, this observation may be substrate dependent.