PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.
Leaves N-glycan core oligosaccharides intact and suitable for further analysis
Non-recombinant with no detectable endoglycosidase F1, F2 or F3 contamination
≥ 95% purity, as determined by SDS-PAGE and intact ESI-MS
Stored in 50% glycerol
Optimal activity and stability for up to 24 months
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: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.
Glycosidase Recognition Site
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
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 %
Properties & 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
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.
To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
PNGase F will not cleave N-linked glycans containing core α1-3 Fucose.
Typical reaction conditions: Please see Protocols
References
Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
Tretter, V. et al. (1991). Eur. J. Biochem.. 199, 647-652.
Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.
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
Citations & Technical Literature
Citations
Product Citation Tool
Additional Citations
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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 Sci Rep; 5, 11603. PubMedID: 26053048, DOI: 10.1038/srep11603
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 Sci Rep; 5, 11603. PubMedID: 26075387, DOI: 10.1038/srep11603
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 Mol Biol Cell; 26, 2168-80. PubMedID: 26136220
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 Sci Rep; PubMedID: 26078040, DOI: 10.1038/srep11603
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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
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
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
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
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. Mol Biol Cell; 26, 2168-80. PubMedID: 26112133, DOI: 10.1091/mbc.E15-01-0040
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 Mol Biol Cell; 26, 2168-80. PubMedID: 26111058, DOI: 10.1091/mbc.E15-01-0040
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. Sci Rep; 5, 11603. PubMedID: 26103163, DOI: 10.1038/srep11603
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
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
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 Sci Rep; PubMedID: 26163436, DOI: 10.1038/srep11603
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
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 Sci Rep; PubMedID: 26083625, DOI: 10.1038/srep11603
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
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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
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