PNGase F

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cloned at neb incubation temp heat inactivation
Catalog #SizeConcentrationPriceQtyAdd to Cart
P0704S15,000 units500,000 units/ml$160.00Add to Cart
P0704L75,000 units500,000 units/ml$640.00Add to Cart
Proteome Analysis
Biosynthesis of Glycans in Eukaryotes,
Glycoprotein Production in Various Expression Systems,



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: Detailed Specificity:
PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α1-3 Fucose residue. This modification is most commonly found in plant and some insect glycoproteins.

Product Source

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

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Glycoprotein Denaturing Buffer-2010X
GlycoBuffer 2-2010X

Advantages and Features


Glycoprotein analysis

  • Removal of high mannose, hybrid, and complex N-glycans from glycoproteins
  • Free of contaminants (Endo F, proteases, etc.)

Properties and Usage

Unit Definition

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

Storage Temperature


Storage Conditions

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

Heat Inactivation

75°C for 10 min

Molecular Weight

Apparent: 36000 daltons


  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


  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.


  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. 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?
  11. What is a good endoglycosidase substrate?
  12. Do detergents inhibit exoglycosidases/endoglycosidases?
  13. What are glycosidases and their uses?

Tech Tips

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 α1-3 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


  1. PNGase F Protocol


The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at or fill out the Technical Support Form for appropriate document.

Feature Articles

Usage Guidelines & Tips

Interactive Tools

Application Notes

NEB Publications

  • Magnelli P, Bielik A, Guthrie E.  (2012). Identification and characterization of protein glycosylation using specific endo- and exoglycosidases Methods Mol Biol. . 801, 189-211. PubMedID: 21987255


  • Holemans T., Sørensen DM., Veen S., Martin S., Hermans D., Kemmer GC., Haute C., Baekelandt V., Pomorski TG., Agostinis P., Wuytack F., Palmgren M., Eggermont J., Vangheluwe P. (2015). A lipid switch unlocks Parkinson's disease-associated ATP13A2 Proc Natl Acad Sci U S A. 112, 9040-5. PubMedID: 26134396
  • AlSalmi W., Mahalingam M., Ananthaswamy N., Hamlin C., Flores D., Gao G., Rao V.B. (2015). A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS. J Biol Chem. 290, 19780-95. PubMedID: 26088135
  • Noble GP., Wang DW., Walsh DJ., Barone JR., Miller MB., Nishina KA., Li S., Supattapone S. (2015). A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers PLoS Pathog. 11, e0127022. PubMedID: 26125623, DOI: 10.1371/journal.ppat.1005017
  • Olsen AL., Lai Y., Dalmau J., Scherer SS., Lancaster E. (2015). Caspr2 autoantibodies target multiple epitopes. Neurol Neuroimmunol Neuroinflamm. 2, e127. PubMedID: 26185774, DOI: 10.1212/NXI.0000000000000127
  • Shakiba N., White C.A., Lipsitz Y.Y., Yachie-Kinoshita A., Tonge P.D., Hussein S.M.I., Puri M.C., Elbaz J., Morrissey-Scoot J., Li M., Munoz J., Benevento M., Rogers I.M., Hanna J.H., Heck A.J.R., Wollscheid B., Nagy A., Zandstra P.W. (2015). CD24 tracks divergent pluripotent states in mouse and human cells. Nat Commun. 6, 7329. PubMedID: 26076835
  • Pritchard L.K., Harvey D.J., Bonomelli C., Crispin M., Doores K.J. (2015). Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope. J Virol. 89, 8932-44. PubMedID: 26085151
  • Go E.P., Herschhorn A., Gu C., Castillo-Menendez L., Zhang S., Mao Y., Chen H., Ding H., Wakefield J.K., Hua D., Liao H.X., Kappes J.C., Sodroski J., Desaire H. (2015). Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140. J Virol. 89, 8245-57. PubMedID: 26018173
  • Liu H., Zou X., Li T., Wang X., Yuan W., Chen Y., Han W. (2015). Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection J Biosci Bioeng. PubMedID: 26140918, DOI: 10.1016/j.jbiosc.2015.05.016
  • Wang L., Zhang X., Pang N., Xiao L., Li Y., Chen N., Ren M., Deng X., Wu J. (2015). Glycation of vitronectin inhibits VEGF-induced angiogenesis by uncoupling VEGF receptor-2-αvβ3 integrin cross-talk Cell Death Dis. 6, e1796. PubMedID: 26111058, DOI: 10.1038/cddis.2015.174
  • Orizio F., Damiati E., Giacopuzzi E., Benaglia G., Pianta S., Schauer R., Schwartz-Albiez R., Borsani G., Bresciani R., Monti E. (2015). Human sialic acid acetyl esterase: Towards a better understanding of a puzzling enzyme. Glycobiology. 25, 992-1006. PubMedID: 26022516
  • Haramoto Y., Takahashi S., Oshima T., Onuma Y., Ito Y., Asashima M. (2015). Insulin-like factor regulates neural induction through an IGF1 receptor-independent mechanism. Sci Rep. 5, 11603. PubMedID: 26112133, DOI: 10.1038/srep11603
  • Julien M., Chauvet S., Scheckenbach KE., Alfaidy N., Chanson M., Benharouga M. (2015). Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability Biochim Biophys Acta. PubMedID: 26083625, DOI: 10.1016/j.bbamcr.2015.06.003
  • Spelios MG., Olsen JA., Kenna LA., Akirav EM. (2015). Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells J Biol Chem. 290, 15250-9. PubMedID: 25911095, DOI: 10.1074/jbc.M114.628784
  • Brown EP., Normandin E., Osei-Owusu NY., Mahan AE., Chan YN., Lai JI., Vaccari M., Rao M., Franchini G., Alter G., Ackerman ME. (2015). Microscale purification of antigen-specific antibodies J Immunol Methods. PubMedID: 26078040, DOI: 10.1016/j.jim.2015.06.005
  • Wang J., Hilchey SP., Hyrien O., Huertas N., Perry S., Ramanunninair M., Bucher D., Zand MS. (2015). Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza. PLoS One. 10, e0127022. PubMedID: 26103163, DOI: 10.1371/journal.pone.0129858
  • Yang W., Zhang Y., Zhou X., Zhang W., Xu X., Chen R., Meng Q., Yuan J., Yang P., Yao B. (2015). Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing PLoS One. 10, e0129294. PubMedID: 26053048, DOI: 10.1371/journal.pone.0129294
  • Netsirisawan P., Chokchaichamnankit D., Srisomsap C., Svasti J., Champattanachai V. (2015). Proteomic Analysis Reveals Aberrant O-GlcNAcylation of Extracellular Proteins from Breast Cancer Cell Secretion Cancer Genomics Proteomics. 12, 201-209. PubMedID: 26136220
  • Chen J., Fang M., Zhao YP., Yi CH., Ji J., Cheng C., Wang MM., Gu X., Sun QS., Chen XL., Gao CF. (2015). Serum N-Glycans: A New Diagnostic Biomarker for Light Chain Multiple Myeloma PLoS One. 10, e0127022. PubMedID: 26075387, DOI: 10.1371/journal.pone.0127022
  • Asazuma HM., Sohn BH., Kim Y.S., Kuo CW., Khoo KH., Kucharski CA., Fraser MJ., Jarvis DL. (2015). Targeted Glycoengineering Extends the Protein N-glycosylation Pathway in the Silkworm Silk Gland Insect Biochem Mol Biol. PubMedID: 26163436, DOI: 10.1016/j.ibmb.2015.07.004
  • Beata O., Jarząb A., Kratz E., Zimmer M., Gamian A., Ferens-Sieczkowska M. (2015). Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors Int J Mol Sci. 16, 14933-50. PubMedID: 26147424, DOI: 10.3390/ijms160714933
  • Malsburg K., Shao S., Hegde RS. (2015). The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon Mol Biol Cell. 26, 2168-80. PubMedID: 25877867, DOI: 10.1091/mbc.E15-01-0040
  • Singh S., Kuntal P., Yadav J., Tang H., Partyka K., Kletter D., Hsueh P., Ensink E., Kc B., Hostetter G., Xu E.H., Bern M., Smith D.F., Mehta A.S., Brand R., Melcher K., Haab B.B. (2015). Upregulation of glycans containing 3' fucose in a subset of pancreatic cancers uncovered using fusion-tagged lectins. J Proteome Res. 14, 2594-605. PubMedID: 25938165
  • Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S (2014). A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells PLoS One. 9(5), e96635. PubMedID: 24804975, DOI: 10.1371/journal.pone.0096635
  • Kwon HM, Lee KH, Han BW, Han MR, Kim DH, Kim DE (2014). An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells PLoS One. 9(5), e97574. PubMedID: 24835440, DOI: 10.1371/journal.pone.0097574
  • Haller G, Li P, Esch C, Hsu S, Goate AM, Steinbach JH (2014). Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior PLoS One. 9(5), e96753. PubMedID: 24804708, DOI: 10.1371/journal.pone.0096753
  • Wright CR, Brown EL, Della-Gatta PA, Ward AC, Lynch GS, Russell AP (2014). G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle Front Physiol. 5, 170. PubMedID: 24822049, DOI: 10.3389/fphys.2014.00170
  • Wicht O, Burkard C, de Haan CA, van Kuppeveld FJ, Rottier PJ, Bosch BJ (2014). Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell J Virol. 88(9), 4943-52. PubMedID: 24554652, DOI: 10.1128/JVI.03451-13
  • Rosenbaek LL, Kortenoeven ML, Aroankins TS, Fenton RA (2014). Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis J Biol Chem. 289(19), 13347-61. PubMedID: 24668812, DOI: 10.1074/jbc.M113.543710
  • Botto L, Cunati D, Coco S, Sesana S, Bulbarelli A, Biasini E, Colombo L, Negro A, Chiesa R, Masserini M, Palestini P (2014). Role of lipid rafts and GM1 in the segregation and processing of prion protein PLoS One. 9(5), e98344. PubMedID: 24859148, DOI: 10.1371/journal.pone.0098344
  • Itahana Y, Han R, Barbier S, Lei Z, Rozen S, Itahana K (2014). The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense Oncogene. PubMedID: 24858040, DOI: 10.1038/onc.2014.119
  • Rosenbaum EE, Vasiljevic E, Brehm KS, Colley NJ (2014). Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster PLoS Genet. 10(5), e1004349. PubMedID: 24785692, DOI: 10.1371/journal.pgen.1004349
  • Möykkynen T, Coleman SK, Semenov A, Keinänen K (2014). The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization J Biol Chem. 289(19), 13197-205. PubMedID: 24652293, DOI: 10.1074/jbc.M113.526301
  • Arakel EC, Brandenburg S, Uchida K, Zhang H, Lin YW, Kohl T, Schrul B, Sulkin MS, Efimov IR, Nichols CG, Lehnart SE, Schwappach B (2014). Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes J Cell Sci. 127(Pt 9), 2106-19. PubMedID: 24569881, DOI: 10.1242/jcs.141440

Quality Control

Quality Assurance Statement

  • No contaminating exoglycosidase or Endoglycosidase F1, F2 or F3 activity could be detected. No contaminating proteolytic activity could be detected.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Glycosidase Activity (TLC):
    The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
  • Protease Activity (SDS-PAGE):
    The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]


The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.


The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at or fill out the Technical Support Form for appropriate document.