N glycosidase f

Manufactured by New England Biolabs

Sourced in United States

N-glycosidase F is an enzyme that cleaves the bond between the asparagine residue and the first N-acetylglucosamine of N-linked glycans. It is commonly used in the analysis of glycoproteins.

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Lab products found in correlation

Pngase f, by New England Biolabs (4 mentions) Endo h, by New England Biolabs (3 mentions) Neuraminidase, by Merck Group (2 mentions) Chloroform, by Merck Group (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Subcellular protein fractionation kit, by Thermo Fisher Scientific (2 mentions) Chop gadd153, by Cell Signaling Technology (1 mentions) Cleaved parp c parp, by Cell Signaling Technology (1 mentions) Anti gapdh antibody, by GeneTex (1 mentions) P erk, by Cell Signaling Technology (1 mentions) Acquity beh amide column, by Waters Corporation (1 mentions) Empower 3, by Waters Corporation (1 mentions) Disuccinimidyl suberate, by Merck Group (1 mentions) Anti ha, by Thermo Fisher Scientific (1 mentions) Supersignal west femto enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Anti total src, by Cell Signaling Technology (1 mentions) Ecl detection reagent, by Cytiva (1 mentions) β actin, by Merck Group (1 mentions) Laemmli buffer, by Thermo Fisher Scientific (1 mentions) Sc 9046, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Peptide-N-glycosidase F (PNGase F) (76 citations) Peptide N-glycosidase F (70 citations) N-glycosidase F (PNGase F) (21 citations) Peptide:N-Glycosidase F (PNGase) (9 citations) Glycerol-free peptidyl-N-glycosidase F (PNGase F) (3 citations) Protein N glycosidase F (2 citations) Glycerol-free Peptide:N-glycosidase F (2 citations) Peptide: N-Glycosidase F (PNGase F) kit (2 citations) Peptide N-Glycosidase F (PNGase F) deglycosylation assay (2 citations) Rapid Peptide-N-Glycosidase (PNGase F), (2 citations)

20 protocols using n glycosidase f

N-Glycosidase F Deglycosylation Protocol

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One milligram of each mAb referred to in the paper was treated with 5,000 U of N-Glycosidase F (also known as PNGase F, NEW ENGLAND Biolabs) in 50 mM ammonium bicarbonate, pH 8.0, at 37˚C for 24 h. The N-glycan removal efficiency was performed with reduced CE-SDS and the treated samples were used for Western Blots, ELISA, or BIAcore and icIEF analysis, as described below.

Yu C., Gao K., Zhu L., Wang W., Wang L., Zhang F., Liu C., Li M., Wormald M.R., Rudd P.M, & Wang J. (2016). At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody. Scientific Reports, 7, 20029.

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Antibody Validation for ER Stress Response

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Antibodies against Lipocalin 2 (LCN2) (#44058), cleaved PARP (cPARP) (#5625), CHOP/GADD153 (#2895), PDI (#3501) and PERK (#3192) were purchased from Cell Signaling Technology. Mouse monoclonal antibodies against BiP/GRP78 (#610978) were from BD Biosciences. Anti-FLAG (#014–22383) monoclonal antibody, holo-transferrin (holo-Tf), Brefeldin A, and ferritic chloride (FeCl₃) were from Fujifilm WAKO Pure Chemical Corporation. As an internal control of E. coli, E. coli RNA polymerase alpha (RNAPα) monoclonal antibody (#WP003) was obtained from BioLegend. Anti-GAPDH antibody was from GeneTex. Anti-SubAB and Stx2 antibodies were prepared as described previously^{70 (link)}. 2,2′-Dipyridyl (DPI) and Deferoxamine mesylate salt (DF) were from Sigma Aldrich; and recombinant human LCN2 (1757-LC-050) was from R&D systems. N-Glycosidase F (P0704S) was purchased from New England BioLabs.

Yahiro K., Ogura K., Goto Y., Iyoda S., Kobayashi T., Takeuchi H., Ohnishi M, & Moss J. (2020). Subtilase cytotoxin induces a novel form of Lipocalin 2, which promotes Shiga-toxigenic Escherichia coli survival. Scientific Reports, 10, 18943.

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Glycan Profiling of HIV-1 Envelope Protein

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Glycan profiling was done as previously described^{18 (link)}. In short, SOSIP trimers were resolved by SDS-PAGE. Bands corresponding to gp140 were excised and the N-linked glycans attached to gp140 protein were released by N-glycosidase F (PNGase F; NEB). Glycans were labeled with 2-aminobenzoic acid. Fluorescently labeled glycans were resolved by HILIC-UPLC using a 2.1 × 10 mm² Acquity BEH Amide Column (1.7 μm particle size) (Waters, Elstree, UK). Fluorescence was measured using an excitation wavelength of 250 nm and a detection wavelength of 428 nm. Data processing was performed using the Empower 3 software (Waters Corporation, Milford, MA, USA). The percentage abundance of oligomannose-type glycans was calculated by integration of the relevant peak areas before and after Endoglycosidase H (EndoH) digestion, following normalization.

Sliepen K., Han B.W., Bontjer I., Mooij P., Garces F., Behrens A.J., Rantalainen K., Kumar S., Sarkar A., Brouwer P.J., Hua Y., Tolazzi M., Schermer E., Torres J.L., Ozorowski G., van der Woude P., de la Peña A.T., van Breemen M.J., Camacho-Sánchez J.M., Burger J.A., Medina-Ramírez M., González N., Alcami J., LaBranche C., Scarlatti G., van Gils M.J., Crispin M., Montefiori D.C., Ward A.B., Koopman G., Moore J.P., Shattock R.J., Bogers W.M., Wilson I.A, & Sanders R.W. (2019). Structure and immunogenicity of a stabilized HIV-1 envelope trimer based on a group-M consensus sequence. Nature Communications, 10, 2355.

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Deglycosylation of Cell Lysates

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Peptide: N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) were obtained from New England Biolabs (Ipswich, MA, USA). Cell lysates were prepared as described above. Cell lysates (20 μg protein) were heated at 100 °C for 10 min and then incubated with or without PNGase F (250 units) or Endo H (250 units) at 37 °C for 60 min according to the manufacturers’ protocols. Proteins were separated by SDS–polyacrylamide gel electrophoresis and analyzed by Western blotting.

Mitsuda S., Yokomichi T., Yokoigawa J, & Kataoka T. (2014). Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum. FEBS Open Bio, 4, 229-239.

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Western Blot Analysis of Protein Tags

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Medium and cell lysates were subjected to SDS-PAGE. Membranes were blocked with fat-free milk (5%) with Tween (1%) for 1 hour and incubated overnight at 4°C in the presence of either a monoclonal mouse anti-V5 (Sigma-Aldrich) or anti-HA (Invitrogen) antibody. Blots were visualized using a goat antimouse horseradish peroxidase-conjugated antibody and SuperSignal West Femto enhanced chemiluminescent substrate (Thermo Scientific) according to manufacturer's instructions. Conditioned media were deglycosylated using N-glycosidase F (New England Biolabs) or covalently cross-linked with disuccinimidyl suberate (Sigma-Aldrich), according to manufacturer's instructions, and the products analyzed by Western blotting.

Bassett J.H., van der Spek A., Logan J.G., Gogakos A., Bagchi-Chakraborty J., Murphy E., van Zeijl C., Down J., Croucher P.I., Boyde A., Boelen A, & Williams G.R. (2015). Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development. Endocrinology, 156(9), 3098-3113.

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Western Blotting Analysis of EMP2 in Breast Cancer

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Preparation of breast cancer cell lines or tissue lysates for western blotting has been described previously (27 ). Cells were lysed in Laemmli buffer, and for EMP2 detection, were treated with N-glycosidase F (New England Biolabs, Beverly, MA) to remove N-link glycosylation (15 (link)). Proteins were separated using SDS-PAGE, transferred onto nitrocellulose membrane and blocked in 10% nonfat dry milk in TBS-Tween-20 buffer. Blots were probed using rabbit anti-human EMP2 antisera (1:2000) (28 (link)). Proteins were then detected by using a horseradish peroxidase conjugated goat anti-rabbit antibody. As a loading control, β-actin expression was detected using primary monoclonal anti-β actin (Sigma) and secondary horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, Piscataway, NJ). Bands were visualized using ECL detection reagents (Amersham).
In some experiments, MDA-MB-468 cells were treated for two hours with 100µg/ml of anti-EMP2 IgG1 or control IgG. Cells were then plated to activate FAK and Src and then lysed after 12 hrs (29 (link), 30 (link)). Separated proteins were probed using anti-576/577p-FAK (Santa Cruz Biotechnology), anti- total FAK (BD Biosciences), anti-416 p-Src (Cell Signaling, Danvers, MA), anti-total Src (Cell Signaling) or β-actin (Sigma-Aldrich).

Fu M., Maresh E.L., Helguera G.F., Kiyohara M., Qin Y., Ashki N., Daniels-Wells T.R., Aziz N., Gordon L.K., Braun J., Elshimali Y., Soslow R.A., Penichet M.L., Goodglick L, & Wadehra M. (2014). Rationale and pre-clinical efficacy of a novel anti-EMP2 antibody for the treatment of invasive breast cancer. Molecular cancer therapeutics, 13(4), 902-915.

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CXCR4 Expression Analysis in MSCs

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MSCs were incubated with IRBs for 2 h and then washed twice with PBS. Fresh medium was utilized for different time periods. After collecting the cells, CXCR4 expression of MSCs was evaluated by fluorescence-activated cell sorting and western blotting.
Cells (>10⁵) and olfactory tissue were lysed in Laemmli buffer according to the manufacturer’s instructions (Invitrogen) and separated on a 12% sodium dodecyl sulfate polyacrylamide gel. Membranes were hybridized with rabbit anti-human CXCR4 at 1:1000 (sc-9046; Santa Cruz Biotechnology, Dallas, TX, USA), with SDF-1 (ab18919; Abcam, Cambridge, UK), or anti-β-actin (1:500; Sigma, St. Louis, MO, USA). Anti-rabbit horseradish peroxidase secondary antibody was used at 1:1000 (Dako, Glostrup, Denmark). Deglycosylation was carried out using N-glycosidase F according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA).
To analyze CXCR4 expression in different subcellular protein fractions, MSCs were incubated with IRBs (108 μM) for 2 h. The cells were cultured for 22 h and the medium was replaced. After cell collection, the proteins expressed in cytoplasm, membrane, and nucleus were separated with a subcellular protein fractionation kit (Thermo Scientific, Waltham, MA, USA).

Yun W.S., Choi J.S., Ju H.M., Kim M.H., Choi S.J., Oh E.S., Seo Y.J, & Key J. (2018). Enhanced Homing Technique of Mesenchymal Stem Cells Using Iron Oxide Nanoparticles by Magnetic Attraction in Olfactory-Injured Mouse Models. International Journal of Molecular Sciences, 19(5), 1376.

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Deglycosylation of Platelet Membrane Proteins

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Briefly, 40 μg membrane fractions from human platelets were heat treated in 30 μL aliquot containing 0.5% SDS (sodium dodecyl sulfate) and 40 mmol/L dithiothreitol at 75°C for 15 min. Then the samples were cooled to room temperature, then reacted with N‐glycosidase F (3000 units; New England Biolabs, Ipswich, MA), 1% NP‐40 for 1 h at 37°C according to the manufacturer's manual. Then, the samples were dissociated with SDS sample buffer and analyzed with polyacrylamide gel electrophoresis in the presence of SDS and the Western blotting.

Hiasa M., Togawa N., Miyaji T., Omote H., Yamamoto A, & Moriyama Y. (2014). Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets. Physiological Reports, 2(6), e12034.

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Glycoproteomic Analysis of HeLa Cells

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Acetonitrile was obtained from Merck (Darmstadt, Germany), while N-glycosidase F (PNGase F, 500 U/μL) was obtained from New England Biolabs (Ipswich, USA). Trypsin was sourced from Beijing Shengxia Proteins Scientific Ltd. (Beijing, China). The bicinchoninic acid protein assay kit and HeLa protein digest standard were acquired from Pierce (Rockford, USA). Unless specified otherwise, all other chemical reagents were obtained from Sigma-Aldrich (St Louis, USA). Distilled water was purified using a Milli-Q system (Millipore, Milford, USA). Ultrafiltration tubes (MWCO=10 kDa) were purchased from Millipore (Amicon Ultra; Merck), and MonoSpin C18 was obtained from Shimadzu (Kyoto, Japan). Affi-Gel boronate gel was sourced from Bio-Rad Laboratories (Hercules, USA).

Ren X., Wu L., Zhang L., Liu Y., Wang G, & Lu H. (2023). Discovery of age-related early-stage glycated proteins based on deep quantitative serum glycated proteome analysis: Age-related changes in the early-glycated proteome. Acta Biochimica et Biophysica Sinica, 55(10), 1659-1667.

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Haptoglobin Glycan Analysis via PNGase F

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N-Glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA). Neuraminidase, sodium hydroxide, methyl iodide, β-mercaptoethanol, chloroform, dimethyl sulfoxide (DMSO), HPLC-grade acetonitrile (ACN), and water and the 96-well vacuum manifold were purchased from Sigma-Aldrich (St. Louis, MO). The MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), the UltraLink hydrazide resin, the Zeba Spin Desalting column, and the SPE 96-well plate packed with 100% porous graphitic carbon (PGC) were purchased from Thermo Scientific (Rockford, IL). The 96-well SpinColumn plate was purchased from Harvard Apparatus (Holliston, MA). Antihuman haptoglobin antibody (CatLog No. ab13429) and a human haptoglobin standard were purchased from Abcam (Cambridge, MA). The empty PEEK column was purchased from MicroSolv Technology Corp. (Eatontown, NJ).

Zhu J., Wu J., Yin H., Marrero J, & Lubman D.M. (2015). Mass Spectrometric N-Glycan Analysis of Haptoglobin from Patient Serum Samples Using a 96-Well Plate Format. Journal of proteome research, 14(11), 4932-4939.

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