产品信息

PNGase A is a recombinant amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and short complex oligosaccharides such as those found in plant and insect cells from N-linked glycoproteins and glycopeptides. PNGase A differs from PNGase F in that it cleaves N-linked glycans with or without α(1,3)-linked core fucose residues.

Substrate Specificity

产品来源

Cloned from Oryza sativa (rice) and expressed in Pichia pastoris.

产品类别:

Endoglycosidases Products,

Proteome Analysis Products

应用:

Glycomics and glycoproteomics,

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
  P0707S     4       PNGase A P0707SVIAL 4 1 x 0.03 ml 5,000 units/ml   GlycoBuffer 3 B1720SVIAL -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 %
  P0707L     4       PNGase A P0707LVIAL 4 1 x 0.15 ml 5,000 units/ml   GlycoBuffer 3 B1720SVIAL -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 > 95% of the carbohydrate from 1 µg of denatured recombinant Avidin produced in Maize in 1 hour at 37°C in a total reaction volume of 10 µl.

  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

  65°C for 10 minutes

  分子量

  实际: 63.8 kDa

  单位活性检测条件

  1 μg of recombinant Avidin is denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 3, two-fold dilutions of PNGase A are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.

• 相关产品

  相关产品

  • p0733-o-glycosidase
  • 蛋白去糖基化混合液 II
  • p0705-pngase-f-glycerol-free
  • Rapid 快速 PNGase F
  • p0711-rapid-pngase-f-non-reducing-format

• 注意事项

  1. PNGase A is active on both glycoproteins and glycopeptides.
  2. PNGase A cannot cleave larger N-glycans such as those from Fetuin, Fibrinogen, IgG, Lactoferrin and Transferrin.
  3. PNGase A is able to cleave high mannose N-glycan structures from Man 3 up to Man 9.
  4. Reactions may be scaled-up linearly to accommodate larger reaction volumes.

操作说明、说明书 & 用法

• 操作说明

  1. Reaction Conditions for PNGase A (P0707)

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 and PNGase A?
  2. Can PNGase A be used under non-denaturing (native) conditions?
  3. Which high mannose structures can PNGase A cleave?
  4. Can PNGase A cleave large, complex oligosaccharides?
  5. What happens to the asparagine after PNGase A removes the sugar?
  6. What is a good PNGase A substrate?
  7. Is PNGase A compatible with downstream analysis such as HPLC and Mass Spectrometry?
  8. How much PNGase A should I use to remove my carbohydrate under native or DTT denaturing conditions?
  9. Why is my protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein. Can a protease inhibitor cocktail be used in a PNGase A reaction?
  10. What are Glycosidases and their uses?

• 实验技巧

  NEB’s version of PNGase A can cleave glycoproteins, there is no need for tryptic digest prior to deglycosylation.

  PNGase A can cleave N-linked glycans containing core α1-3 Fucose.

  PNGase A activity is inhibited by SDS, therefore under denaturing conditions it is essential to have NP-40 present in the reaction mixture in a 1:1 ratio.

  A good positive control substrate is recombinant avidin from maize or HRP.