产品信息

Peptide: N-Glycosidase F, also known as PNGase F, is a recombinant amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1).

产品来源

Cloned from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) and expressed in E. coli (2).

特异性

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
  P0708S     -20       PNGase F, Recombinant P0708SVIAL -20 1 x 0.03 ml 500,000 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 %
  P0708L     -20       PNGase F, Recombinant P0708LVIAL -20 1 x 0.15 ml 500,000 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 > 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.

  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 NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F 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

  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

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

  热失活

  75°C for 10 minutes

  分子量

  实际: 36000 daltons

• 优势和特性

  应用特性

  • Removal of carbohydrate residues from proteins

• 相关产品

  相关产品

  • RNase B（对照底物）
  • 糖苷内切酶反应缓冲液套装
  • p0702-endo-h
  • p0703-endo-hf
  • p0706-remove-it-pngase-f
  • p0733-o-glycosidase

• 注意事项

  1. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  2. Since PNGase F, Recombinant activity 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.
  3. PNGase F, Recombinant will not cleave N-linked glycans containing core α1-3 Fucose.

• 参考文献

  1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  2. Chen, M. New England Biolabs, Inc., Unpublished observation

操作说明、说明书 & 用法

• 操作说明

  1. PNGase F Protocol

• 使用指南

  • Detailed Characterization of Several Glycosidase Enzymes
  • Glycobiology Unit Conversion Chart

• 应用实例

  • AppNote_Remove-iT_PNGase_F_Effective_Release_and_Recovery_of_Neutral_and_Sialylated_N-glycans
  • AppNote_Unbiased_and_Fast_IgG_Deglycosylation _for_Accurate_N-glycan_Analysis_using_Rapid_PNGase_F
  • AppNote_Proteomics_Fast_and_Efficient_Antibody_Deglycosylation_using_Rapid_PNGase_F
  • AppNote_Glycan_Analysis_of_Murine_IgG2a_by_Enzymatic_Digestion_with_PNGase_F_and_Trypsin
  • Glycan Analysis of Murine IgG2a by Enzymatic Digestion with PNGase F and Trypsin, Followed by Mass Spectrometric Analysis
  • Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
  • Characterization of Glycans from Erbitux®, Rituxan® and Enbrel® using PNGase F (Glycerol-free), Recombinant
  • Remove iT PNGase F Effective Release and Recovery of Neutral and Sialylated N glycans

工具 & 资源

• 选择指南

  • Endoglycosidase Selection Chart

FAQs & 问题解决指南

• FAQs

  1. Is there a difference between PNGase F (P0704/P0705) and PNGase F, Recombinant (P0708/P0709)?
  2. I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem?
  3. What happens to the asparagine after PNGase removes the sugar?
  4. Why is 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 F, Recombinant reaction?
  5. What is the difference between PNGase F, Endo H and O-Glycosidase?
  6. Does PNGase F work in Urea?
  7. What are the typical reaction conditions for PNGase F, Recombinant?
  8. How much PNGase F, Recombinant should I use to remove my carbohydrate under native or DTT denaturing conditions?
  9. How do I inhibit PNGase F, Recombinant?
  10. Is PNGase F, Recombinant compatible with downstream analysis such as HPLC and Mass Spectrometry?
  11. What are Glycosidases and their uses?
  12. Do detergents inhibit exoglycosidases/endoglycosidases?
  13. 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?
  14. What is a good endoglycosidase substrate?

• 实验技巧

  1. You can use this enzyme under native or denaturing conditions
  2. Under native conditions we recommend adding more enzyme and using longer incubation times
  3. PNGase F 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.
  4. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose (PNGase A must be used in this instance)
  5. A good positive control substrate is RNase B