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PNGase F

Catalog # Concentration Size List Price Quantity Your Price
P0704L 500000 units/ml 75000 units $1,299.00
$1,169.10
P0704S 500000 units/ml 15000 units $326.00
$293.40
Catalog # Size List Price Your Price
P0704L 75000 units $1,299.00
$1,169.10
P0704S 15000 units $326.00
$293.40
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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
  • Can be used under native or denaturing conditions
Featured Videos
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  • Monarch_MiniprepProtocol_VideoThumb
    Overview of Glycobiology

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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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 %
  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 %
Application Features
  • Removal of high mannose N-glycans from glycoproteins

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

1X NP-40
1% NP-40 in MilliQ-H2O

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2
50 mM Sodium Phosphate
(pH 7.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

75°C for 10 minutes

Molecular Weight

Apparent: 36000 daltons


Companion Products
Notes
  • 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.
FAQs
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 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
Additional Citations
  • 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 Sci RepPubMedID: 26140918, DOI: 10.1038/srep11603
  • 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 RepPubMedID: 26078040, DOI: 10.1038/srep11603
  • 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
  • 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
  • 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
  • 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
  • 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 Mol Biol Cell 26, 2168-80.PubMedID: 25911095, DOI: 10.1091/mbc.E15-01-0040
  • 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 RepPubMedID: 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 RepPubMedID: 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
  • 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 Sci Rep 5, 11603.PubMedID: 26125623, DOI: 10.1038/srep11603
  • 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 OncogenePubMedID: 24858040, DOI: 10.1038/onc.2014.119
  • 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
  • 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
  • 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
  • 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
  • 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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