p0704  (New England Biolabs)


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    Name:
    PNGase F native
    Description:
    PNGase F native 75 000 units
    Catalog Number:
    p0704l
    Price:
    754
    Size:
    75 000 units
    Category:
    Glycosidases
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    Structured Review

    New England Biolabs p0704
    PNGase F native
    PNGase F native 75 000 units
    https://www.bioz.com/result/p0704/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p0704 - by Bioz Stars, 2020-09
    99/100 stars

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    Sonication:

    Article Title: Acute cellular uptake of abnormal prion protein is cell type and scrapie strain independent
    Article Snippet: .. PK digested lysates were precipitated in four volumes of cold methanol for 2 hrs at −20°C followed by centrifugation at 20,800 × g for 30 min. Pellets were sonicated into sample buffer (2.5% SDS, 3 mM EDTA, 2% β-mercaptoethanol, 5% glycerol, 0.02% bromphenol blue, and 63 mM Tris-HCL, pH 6.8), boiled for 3 min, optionally PNGaseF treated for 12 hours according to the manufacturer’s instructions (New England Biolabs), and loaded on 16% Tris-Glycine precast gels (Invitrogen). .. PrP was detected by western blot analysis using the mouse monoclonal antibody 3F4 (1:3,000) followed by secondary ECL-anti-mouse IgG (1:5,000) (Amersham) or anti-mouse IR-dye 800CW (1:10,000) (Li-Cor).

    Centrifugation:

    Article Title: Acute cellular uptake of abnormal prion protein is cell type and scrapie strain independent
    Article Snippet: .. PK digested lysates were precipitated in four volumes of cold methanol for 2 hrs at −20°C followed by centrifugation at 20,800 × g for 30 min. Pellets were sonicated into sample buffer (2.5% SDS, 3 mM EDTA, 2% β-mercaptoethanol, 5% glycerol, 0.02% bromphenol blue, and 63 mM Tris-HCL, pH 6.8), boiled for 3 min, optionally PNGaseF treated for 12 hours according to the manufacturer’s instructions (New England Biolabs), and loaded on 16% Tris-Glycine precast gels (Invitrogen). .. PrP was detected by western blot analysis using the mouse monoclonal antibody 3F4 (1:3,000) followed by secondary ECL-anti-mouse IgG (1:5,000) (Amersham) or anti-mouse IR-dye 800CW (1:10,000) (Li-Cor).

    Immunoprecipitation:

    Article Title: Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica ▿
    Article Snippet: .. To examine whether the smear was due to posttranslational modifications such as glycosylation, we treated immunoprecipitated Eh PNT-HA with TFMS or PNGase F. The pattern of immunoblots with anti-HA antibody was not affected, while the apparent molecular mass of control fetuin decreased ( ). ..

    Western Blot:

    Article Title: Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica ▿
    Article Snippet: .. To examine whether the smear was due to posttranslational modifications such as glycosylation, we treated immunoprecipitated Eh PNT-HA with TFMS or PNGase F. The pattern of immunoblots with anti-HA antibody was not affected, while the apparent molecular mass of control fetuin decreased ( ). ..

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    New England Biolabs pngase f
    Effect of N -linked glycosylation on the binding of IL13Rα2 to recombinant IL13Rα2. A , binding of IL13Rα2 to control and Pngase F-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml and treated with native buffer or with 1 milliunit/well <t>Pngase</t> F in native buffer for 3 h at 37 °C. An ELISA for binding of the IL13Rα2 (clone 47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z and rhIL-13 was performed, and the data of one representative experiment from three independent experiments are shown. A paired t test was used to evaluate the difference between control and Pngase F-treated groups ( n = 4). *, p
    Pngase F, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pngase f/product/New England Biolabs
    Average 99 stars, based on 270 article reviews
    Price from $9.99 to $1999.99
    pngase f - by Bioz Stars, 2020-09
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    Effect of N -linked glycosylation on the binding of IL13Rα2 to recombinant IL13Rα2. A , binding of IL13Rα2 to control and Pngase F-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml and treated with native buffer or with 1 milliunit/well Pngase F in native buffer for 3 h at 37 °C. An ELISA for binding of the IL13Rα2 (clone 47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z and rhIL-13 was performed, and the data of one representative experiment from three independent experiments are shown. A paired t test was used to evaluate the difference between control and Pngase F-treated groups ( n = 4). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization and Immunotherapeutic Implications for a Novel Antibody Targeting Interleukin (IL)-13 Receptor ?2 *

    doi: 10.1074/jbc.M112.370015

    Figure Lengend Snippet: Effect of N -linked glycosylation on the binding of IL13Rα2 to recombinant IL13Rα2. A , binding of IL13Rα2 to control and Pngase F-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml and treated with native buffer or with 1 milliunit/well Pngase F in native buffer for 3 h at 37 °C. An ELISA for binding of the IL13Rα2 (clone 47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z and rhIL-13 was performed, and the data of one representative experiment from three independent experiments are shown. A paired t test was used to evaluate the difference between control and Pngase F-treated groups ( n = 4). *, p

    Article Snippet: Goat anti-mouse antibody conjugated with peroxidase was purchased from Chemicon International (Temicula, CA), and Pngase F was purchased from New England Biolabs (Ipswich, MA).

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Clone Assay

    Proteomics analysis of serum glycoproteins from treated and untreated mice. 21 days after cell injection, mice underwent cryo-thermal or no treatment as control. Afterwards, mice were kept for another 2 h, 2, 5, 8, 11, 14, 21 and 28 days. For shotgun proteomics, serum were collected and pooled under each condition. After protein denaturation, reduction and alkylation, pooled sera were then digested to peptides. Subsequently, glycopeptides were captured by the hydrazide solid phase and cleaved by PNGase F enzyme. The released N-glycosylated peptides were than labeled with 8-plex iTRAQ reagent and mixed for subsequent off-gel pre-fractionation. N-glycosylated peptides extracted from healthy mice were labeled as the global reference. Each fraction was then subjected to LC-MS/MS for protein identification and quantification. Database search and analysis were performed in TPP and significant candidates were verified using quantitative PRM method for target analysis.

    Journal: Theranostics

    Article Title: Interleukin-6 Induced “Acute” Phenotypic Microenvironment Promotes Th1 Anti-Tumor Immunity in Cryo-Thermal Therapy Revealed By Shotgun and Parallel Reaction Monitoring Proteomics

    doi: 10.7150/thno.14394

    Figure Lengend Snippet: Proteomics analysis of serum glycoproteins from treated and untreated mice. 21 days after cell injection, mice underwent cryo-thermal or no treatment as control. Afterwards, mice were kept for another 2 h, 2, 5, 8, 11, 14, 21 and 28 days. For shotgun proteomics, serum were collected and pooled under each condition. After protein denaturation, reduction and alkylation, pooled sera were then digested to peptides. Subsequently, glycopeptides were captured by the hydrazide solid phase and cleaved by PNGase F enzyme. The released N-glycosylated peptides were than labeled with 8-plex iTRAQ reagent and mixed for subsequent off-gel pre-fractionation. N-glycosylated peptides extracted from healthy mice were labeled as the global reference. Each fraction was then subjected to LC-MS/MS for protein identification and quantification. Database search and analysis were performed in TPP and significant candidates were verified using quantitative PRM method for target analysis.

    Article Snippet: N-glycopeptides were then released by PNGase F (New England Biolab, Ipswich, MA).

    Techniques: Mouse Assay, Injection, Labeling, Fractionation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    PrP-res 3F4 uptake into neural cells is significantly decreased at 18°C MoL42-CFD5 cells were exposed to infected brain homogenate from 22L(3F4), ME7(3F4) or Obi(3F4) scrapie for 24 hours at either 37°C or 18°C. Samples were PNGaseF treated to remove complex glycans. Non-PK digested infected brain homogenates were run to illustrate total PrP levels (lanes 1,7,13) while PK-digested brain homogenates were run as a positive control for PrP-res (lanes 2,8,14). Some PrP-sen 3F4 was taken up by cells at 37°C (−PK lanes 3, 9, 15) and PrP-res 3F4 was detected in cells which had been exposed to scrapie brain homogenate and incubated at 37°C (+PK lanes 4, 10, 16). Exposure time for Panel A was 8 minutes. After overnight exposures of the gels, some PrP-sen 3F4 (−PK lanes 5, 11, 17) and a low level of PrP-res 3F4 (+PK lanes 6, 12, 18) were detected in cells which had been exposed to scrapie brain homogenate and incubated at 18°C (Panel B). All blots were analyzed using the mouse monoclonal antibody 3F4 and developed using ECL (Amersham).

    Journal: Virology

    Article Title: Acute cellular uptake of abnormal prion protein is cell type and scrapie strain independent

    doi: 10.1016/j.virol.2008.07.006

    Figure Lengend Snippet: PrP-res 3F4 uptake into neural cells is significantly decreased at 18°C MoL42-CFD5 cells were exposed to infected brain homogenate from 22L(3F4), ME7(3F4) or Obi(3F4) scrapie for 24 hours at either 37°C or 18°C. Samples were PNGaseF treated to remove complex glycans. Non-PK digested infected brain homogenates were run to illustrate total PrP levels (lanes 1,7,13) while PK-digested brain homogenates were run as a positive control for PrP-res (lanes 2,8,14). Some PrP-sen 3F4 was taken up by cells at 37°C (−PK lanes 3, 9, 15) and PrP-res 3F4 was detected in cells which had been exposed to scrapie brain homogenate and incubated at 37°C (+PK lanes 4, 10, 16). Exposure time for Panel A was 8 minutes. After overnight exposures of the gels, some PrP-sen 3F4 (−PK lanes 5, 11, 17) and a low level of PrP-res 3F4 (+PK lanes 6, 12, 18) were detected in cells which had been exposed to scrapie brain homogenate and incubated at 18°C (Panel B). All blots were analyzed using the mouse monoclonal antibody 3F4 and developed using ECL (Amersham).

    Article Snippet: PK digested lysates were precipitated in four volumes of cold methanol for 2 hrs at −20°C followed by centrifugation at 20,800 × g for 30 min. Pellets were sonicated into sample buffer (2.5% SDS, 3 mM EDTA, 2% β-mercaptoethanol, 5% glycerol, 0.02% bromphenol blue, and 63 mM Tris-HCL, pH 6.8), boiled for 3 min, optionally PNGaseF treated for 12 hours according to the manufacturer’s instructions (New England Biolabs), and loaded on 16% Tris-Glycine precast gels (Invitrogen).

    Techniques: Infection, Positive Control, Incubation

    Soluble shed MICA (sMICA) in plasma of MICAgen mice does not down-regulate NKG2D and is derived, at least in part, from hematopoietic cells. (A,B) Activated MICAgen splenocytes shed substantial amounts of sMICA. Freshly isolated splenocytes of MICAgen mice were treated in vitro with either lipopolysaccharide (LPS) or PMA plus ionomycin (PMA/I), or left untreated (NT) for various times. (A) Concentrations of sMICA in the respective culture supernatants were determined by a MICA-specific ELISA. Data from stimulations of splenocytes from three different mice are shown with mean and standard deviation. ND, not detectable. (B) Detection of sMICA in PNGaseF-digested culture supernatants of activated splenocytes by immunoblotting using mAb BAMO1. For comparison, lysates of PMA/I-stimulated splenocytes (full-length MICA) and supernatants of B16F10-MICA transductants (sMICA) were analyzed. (C) Plasma of MICAgen mice contains substantial amounts (~0.4 ng/ml) of sMICA but markedly less than H2-K b -MICA mice (~75 ng/ml). Plasma of nontgLM, as well as of MICAgen and H2-K b -MICA mice, was analyzed for sMICA by ELISA. Each symbol represents sMICA levels of an individual mouse. ND, not detectable. Mean and standard deviation are shown. (D,E) NKG2D surface expression on NK cells after (E) direct co-culture with splenocytes from MICAgen, or H2-K b -MICA mice, or nontgLM, respectively, or (D) co-culture with such splenocytes in a transwell setting. Carboxyfluorescein succinimidyl ester (CFSE) pre-labeled splenic NK cells from nontgLM (D) placed in permeable transwell inserts into cultures of splenocytes from nontgLM, or MICAgen, or H2-K b -MICA mice (E) or directly co-cultured with splenocytes from nontgLM, or MICAgen, or H2-K b -MICA mice, respectively. After 24 h culture, NK cells were stained with anti-NKG2D mAb and analyzed by flow cytometry with gates set on CFSE + CD3 − NK1.1 + cells. Representative stainings of NK cells co-cultured with splenocytes from nontgLM (black line), MICAgen (red line), and H2-K b -MICA (gray line) were overlayed. NKG2D expression of NK cells cultured without splenocytes (blue line) is shown for control. Isotype controls stainings (rat IgG1k-BV421) of NK cells co-cultured with MICAgen splenocytes also overlayed. Mean and standard deviation are shown. Each dot represents an individual mouse. One-way ANOVA test was performed (**** p

    Journal: Frontiers in Immunology

    Article Title: MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA

    doi: 10.3389/fimmu.2020.00960

    Figure Lengend Snippet: Soluble shed MICA (sMICA) in plasma of MICAgen mice does not down-regulate NKG2D and is derived, at least in part, from hematopoietic cells. (A,B) Activated MICAgen splenocytes shed substantial amounts of sMICA. Freshly isolated splenocytes of MICAgen mice were treated in vitro with either lipopolysaccharide (LPS) or PMA plus ionomycin (PMA/I), or left untreated (NT) for various times. (A) Concentrations of sMICA in the respective culture supernatants were determined by a MICA-specific ELISA. Data from stimulations of splenocytes from three different mice are shown with mean and standard deviation. ND, not detectable. (B) Detection of sMICA in PNGaseF-digested culture supernatants of activated splenocytes by immunoblotting using mAb BAMO1. For comparison, lysates of PMA/I-stimulated splenocytes (full-length MICA) and supernatants of B16F10-MICA transductants (sMICA) were analyzed. (C) Plasma of MICAgen mice contains substantial amounts (~0.4 ng/ml) of sMICA but markedly less than H2-K b -MICA mice (~75 ng/ml). Plasma of nontgLM, as well as of MICAgen and H2-K b -MICA mice, was analyzed for sMICA by ELISA. Each symbol represents sMICA levels of an individual mouse. ND, not detectable. Mean and standard deviation are shown. (D,E) NKG2D surface expression on NK cells after (E) direct co-culture with splenocytes from MICAgen, or H2-K b -MICA mice, or nontgLM, respectively, or (D) co-culture with such splenocytes in a transwell setting. Carboxyfluorescein succinimidyl ester (CFSE) pre-labeled splenic NK cells from nontgLM (D) placed in permeable transwell inserts into cultures of splenocytes from nontgLM, or MICAgen, or H2-K b -MICA mice (E) or directly co-cultured with splenocytes from nontgLM, or MICAgen, or H2-K b -MICA mice, respectively. After 24 h culture, NK cells were stained with anti-NKG2D mAb and analyzed by flow cytometry with gates set on CFSE + CD3 − NK1.1 + cells. Representative stainings of NK cells co-cultured with splenocytes from nontgLM (black line), MICAgen (red line), and H2-K b -MICA (gray line) were overlayed. NKG2D expression of NK cells cultured without splenocytes (blue line) is shown for control. Isotype controls stainings (rat IgG1k-BV421) of NK cells co-cultured with MICAgen splenocytes also overlayed. Mean and standard deviation are shown. Each dot represents an individual mouse. One-way ANOVA test was performed (**** p

    Article Snippet: For deglycosylation of proteins, lysates, or culture supernatants were treated with PNGaseF (New England BioLabs, Frankfurt, Germany) according to the manufacturer's instructions for 2 h at 37°C.

    Techniques: Mouse Assay, Derivative Assay, Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation, Expressing, Co-Culture Assay, Labeling, Cell Culture, Staining, Flow Cytometry

    Activation-induced surface expression of MICA molecules on MICAgen splenocytes. (A) MICA is barely detectable on total splenocytes of MICAgen mice in contrast to splenocytes of H2-K b -MICA mice. Freshly isolated splenocytes were stained for surface MICA and assessed flow cytometry. (B) Differential low MICA expression by subsets of MICAgen splenocytes. Freshly isolated splenocytes were stained for surface MICA in addition to various immune markers, and gated for B cells (CD19 + CD3 − ), T cells (CD19 − CD3 + ), NK cells (CD11b + Gr1 − NKp46 + ), and myeloid cells (CD11b + Gr1 + ), respectively. (A,B) MICA stainings (biotinylated BAMO1 plus SA-BV421) of (subgated) splenocytes from MICAgen mice (red line) are overlayed with those of H2-K b -MICA mice (blue line) and nontgLM (black line), and negative control stainings (biotinylated irrelevant mouse IgG1 plus SA-BV421) of MICAgen mice (red filled). (C,D) Strong induction of surface MICA expression by activated MICAgen splenocytes. Freshly isolated splenocytes of MICAgen mice were treated (C) with phorbol myristate acetate (PMA) plus ionomycin (PMA/I) or (D) with lipopolysaccharide (LPS) for various times and subsequently MICA cell surface expression of lymphocyte subsets monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. (C,D) MICA stainings of subgated splenocytes treated for 0 (red line), 8 (green line), and 24 h (blue line) are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment is also overlayed (filled light blue). (E) Activation-induced surface MICA expression on MICAgen splenocytes is transient. Freshly isolated splenocytes of MICAgen mice were cultured in presence of PMA/I for either 0.5 (left) or 2 h (right), extensively washed, and subsequently cultured for up to 96 h. MICA surface expression on B cells monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. MICA stainings of B cells before stimulation with PMA/I (red line), and 24 (blue), 48 (black), 72 (green), or 96 h (gray) after begin of stimulation are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment are also overlayed (filled blue). (F) Activation of MICAgen splenocytes results in de novo induced MICA glycoprotein expression. Freshly isolated splenocytes of nontgLM, MICAgen, and H2-K b -MICA mice were treated for 24 h with LPS or PMA/I or left untreated (NT), and subsequently, PNGaseF-treated cell lysates were analyzed by immunoblotting with biotinylated BAMO1. Detection of actin as loading control.

    Journal: Frontiers in Immunology

    Article Title: MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA

    doi: 10.3389/fimmu.2020.00960

    Figure Lengend Snippet: Activation-induced surface expression of MICA molecules on MICAgen splenocytes. (A) MICA is barely detectable on total splenocytes of MICAgen mice in contrast to splenocytes of H2-K b -MICA mice. Freshly isolated splenocytes were stained for surface MICA and assessed flow cytometry. (B) Differential low MICA expression by subsets of MICAgen splenocytes. Freshly isolated splenocytes were stained for surface MICA in addition to various immune markers, and gated for B cells (CD19 + CD3 − ), T cells (CD19 − CD3 + ), NK cells (CD11b + Gr1 − NKp46 + ), and myeloid cells (CD11b + Gr1 + ), respectively. (A,B) MICA stainings (biotinylated BAMO1 plus SA-BV421) of (subgated) splenocytes from MICAgen mice (red line) are overlayed with those of H2-K b -MICA mice (blue line) and nontgLM (black line), and negative control stainings (biotinylated irrelevant mouse IgG1 plus SA-BV421) of MICAgen mice (red filled). (C,D) Strong induction of surface MICA expression by activated MICAgen splenocytes. Freshly isolated splenocytes of MICAgen mice were treated (C) with phorbol myristate acetate (PMA) plus ionomycin (PMA/I) or (D) with lipopolysaccharide (LPS) for various times and subsequently MICA cell surface expression of lymphocyte subsets monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. (C,D) MICA stainings of subgated splenocytes treated for 0 (red line), 8 (green line), and 24 h (blue line) are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment is also overlayed (filled light blue). (E) Activation-induced surface MICA expression on MICAgen splenocytes is transient. Freshly isolated splenocytes of MICAgen mice were cultured in presence of PMA/I for either 0.5 (left) or 2 h (right), extensively washed, and subsequently cultured for up to 96 h. MICA surface expression on B cells monitored by flow cytometry using biotinylated AMO1 plus SA-BV421. MICA stainings of B cells before stimulation with PMA/I (red line), and 24 (blue), 48 (black), 72 (green), or 96 h (gray) after begin of stimulation are overlayed. Negative control stainings (biotinylated irrelevant IgG1 plus SA-BV421) of samples at 24 h of treatment are also overlayed (filled blue). (F) Activation of MICAgen splenocytes results in de novo induced MICA glycoprotein expression. Freshly isolated splenocytes of nontgLM, MICAgen, and H2-K b -MICA mice were treated for 24 h with LPS or PMA/I or left untreated (NT), and subsequently, PNGaseF-treated cell lysates were analyzed by immunoblotting with biotinylated BAMO1. Detection of actin as loading control.

    Article Snippet: For deglycosylation of proteins, lysates, or culture supernatants were treated with PNGaseF (New England BioLabs, Frankfurt, Germany) according to the manufacturer's instructions for 2 h at 37°C.

    Techniques: Activation Assay, Expressing, Mouse Assay, Isolation, Staining, Flow Cytometry, Negative Control, Cell Culture