产品信息

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

Detailed Specificity:

产品来源

PNGase F is purified from Flavobacterium meningosepticum (3) and it is free of proteases and Endo F activities.

Glycosidase Recognition Site

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
  P0704S     -20       PNGase F P0704SVIAL -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 %
  P0704L     -20       PNGase F P0704LVIAL -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

  20 mM Tris-HCl
  50 mM NaCl
  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 high mannose N-glycans from glycoproteins

• 相关产品

  相关产品

  • p0702-endo-h
  • p0703-endo-hf
  • p0705-pngase-f-glycerol-free
  • RNase B（对照底物）
  • p0733-o-glycosidase
  • p0706-remove-it-pngase-f
  • Endo S
  • 糖苷内切酶 D

• 注意事项

  1. Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture. Why this non-ionic detergent counteracts the SDS inhibition is unknown at present.
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  3. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose.
  4. Typical reaction conditions: Please see Protocols

• 参考文献

  1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  2. Tretter, V. et al. (1991). Eur. J. Biochem.. 199, 647-652.
  3. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.

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_Glycan_Analysis_of_Murine_IgG_by_Enzymatic_Digestion_with_Endo_S_and_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
  • Characterization of Glycans from Erbitux®, Rituxan® and Enbrel® using PNGase F (Glycerol-free), Recombinant
  • Enzymatic removal of N– and O-glycans using PNGase F or the Protein Deglycosylation Mix
  • Glycan Analysis of Murine IgG by Enzymatic Digestion with Endo S and PNGase F Followed by Mass Spectrometric Analysis
  • Remove iT PNGase F Effective Release and Recovery of Neutral and Sialylated N glycans
  • Glycan Analysis of Murine IgG2a by Enzymatic Digestion with PNGase F and Trypsin, Followed by Mass Spectrometric Analysis
  • A Fast One-Step Digestion of DNA or RNA for Global Detection and Characterization of Nucleotide Modifications

工具 & 资源

• 选择指南

  • Endoglycosidase Selection Chart

FAQs & 问题解决指南

• FAQs

  1. Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
  2. What happens to the asparagine after PNGase removes the sugar?
  3. Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
  4. What are the typical reaction conditions for PNGase F?
  5. Does PNGase F work in Urea?
  6. How do I inhibit PNGase F?
  7. How much PNGase F should I use to remove my carbohydrate under native conditions?
  8. I tried the PNGase F on my glycoprotein and didn’t see removal of the carbohydrate. What could be the problem?
  9. What is the difference between PNGase F, Endo H and O-Glycosidase?
  10. Do detergents inhibit exoglycosidases/endoglycosidases?
  11. 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?
  12. What are Glycosidases and their uses?
  13. What is a good endoglycosidase substrate?

• 实验技巧

  PNGase F
  You can use this enzyme under native or denaturing conditions
  Under native conditions we recommend adding more enzyme and using longer incubation times
  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.
  PNGase F will not cleave N-linked glycans containing core a1-3 Fucose (PNGase A must be used in this instance)
  Enzyme activity varies at different temperatures: 37°C – 100%; 30°C – 100%; 23°C – 65%; 17°C – 40% and 3°C – 0%
  A good positive control substrate is RNase B