endoglycosidase h reaction buffers  (New England Biolabs)


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    Name:
    Endoglycosidase Reaction Buffer Pack
    Description:
    Endoglycosidase Reaction Buffer Pack 4 0 ml
    Catalog Number:
    b0701s
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    23
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    4 0 ml
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    Buffers
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    New England Biolabs endoglycosidase h reaction buffers
    Endoglycosidase Reaction Buffer Pack
    Endoglycosidase Reaction Buffer Pack 4 0 ml
    https://www.bioz.com/result/endoglycosidase h reaction buffers/product/New England Biolabs
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    endoglycosidase h reaction buffers - by Bioz Stars, 2020-01
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    1) Product Images from "Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody"

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody

    Journal: eLife

    doi: 10.7554/eLife.36217

    Expression and functionality of the P2X7-EGFP constructs in HEK cells and expression of the transgene in mice. ( A ) The EGFP sequence was fused via a Strep-tag-His-tag linker to the very C-terminus of the mouse P2X7 sequence. (B/C) Protein extracts from transiently transfected (Lipofectamine 2000, Invitrogen) HEK cells (DSMZ (ACC 305), regularly tested for mycoplasma contamination) were separated by SDS-PAGE with endoglycosidase treatment as indicated. Gels were analyzed by western blotting with a P2X7-specific antibody (Alamone, extracellular) ( B ) or by direct EGFP-fluorescence scanning ( C ). ( D ) Normalized dose–response curves for ATP-induced ethidium uptake. HEK293 cells were cultured and transfected (2 μg DNA/well of a six-well-plate, Lipofectamin, Thermo Fisher Scientific). After 27 hr, cells were seeded in 96-well plates (5 × 10 4 cells/well) and incubated in the presence of 20 μM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( Bruzzone et al., 2010 ). Lines represent nonlinear curve fits of the Hill equation to the data and were normalized to the calculated maximal responses. EC 50 values are 582 (CI 498–681), 840 (CI 644–1098) and 582 (CI 457–740) for wt (L variant) and EGFP-tagged L and P variants, respectively. Error bars represent SEM (n = 4–7). CI = 95% confidence interval. ( E ) Patch-clamp recordings from HEK cells transfected as above with wt and EGFP-tagged P2X7 (L variants). Recordings were performed as described ( Nicke et al., 2009 ) in normal or low divalent cation (DIC) containing extracellular solution to account for possible unspecific effects ( Nörenberg et al., 2016 ). Representative current traces from n > 3 cells are shown. Due to problems with the perfusion, one trace is incomplete (1 mM ATP for P2X7-EGFP in low DIC). ( F ) Southern blot controls for correct modification and integrity of the P2X7 BAC clone during subsequent homologous recombination steps. ( G ) Reproducibility and stability of endogenous and transgenic P2X7 protein expression. Protein extracts from four line 17 mice were separated by SDS-PAGE and quantified by western blotting and infrared imaging using antibodies against P2X7 (Synaptic Systems) and vinculin and fluorescent secondary antibodies. Data are presented as mean ±SD from four individual mice. No significant difference of endogenous P2X7 expression was found in two independent experiments.
    Figure Legend Snippet: Expression and functionality of the P2X7-EGFP constructs in HEK cells and expression of the transgene in mice. ( A ) The EGFP sequence was fused via a Strep-tag-His-tag linker to the very C-terminus of the mouse P2X7 sequence. (B/C) Protein extracts from transiently transfected (Lipofectamine 2000, Invitrogen) HEK cells (DSMZ (ACC 305), regularly tested for mycoplasma contamination) were separated by SDS-PAGE with endoglycosidase treatment as indicated. Gels were analyzed by western blotting with a P2X7-specific antibody (Alamone, extracellular) ( B ) or by direct EGFP-fluorescence scanning ( C ). ( D ) Normalized dose–response curves for ATP-induced ethidium uptake. HEK293 cells were cultured and transfected (2 μg DNA/well of a six-well-plate, Lipofectamin, Thermo Fisher Scientific). After 27 hr, cells were seeded in 96-well plates (5 × 10 4 cells/well) and incubated in the presence of 20 μM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( Bruzzone et al., 2010 ). Lines represent nonlinear curve fits of the Hill equation to the data and were normalized to the calculated maximal responses. EC 50 values are 582 (CI 498–681), 840 (CI 644–1098) and 582 (CI 457–740) for wt (L variant) and EGFP-tagged L and P variants, respectively. Error bars represent SEM (n = 4–7). CI = 95% confidence interval. ( E ) Patch-clamp recordings from HEK cells transfected as above with wt and EGFP-tagged P2X7 (L variants). Recordings were performed as described ( Nicke et al., 2009 ) in normal or low divalent cation (DIC) containing extracellular solution to account for possible unspecific effects ( Nörenberg et al., 2016 ). Representative current traces from n > 3 cells are shown. Due to problems with the perfusion, one trace is incomplete (1 mM ATP for P2X7-EGFP in low DIC). ( F ) Southern blot controls for correct modification and integrity of the P2X7 BAC clone during subsequent homologous recombination steps. ( G ) Reproducibility and stability of endogenous and transgenic P2X7 protein expression. Protein extracts from four line 17 mice were separated by SDS-PAGE and quantified by western blotting and infrared imaging using antibodies against P2X7 (Synaptic Systems) and vinculin and fluorescent secondary antibodies. Data are presented as mean ±SD from four individual mice. No significant difference of endogenous P2X7 expression was found in two independent experiments.

    Techniques Used: Expressing, Construct, Mouse Assay, Sequencing, Strep-tag, Transfection, SDS Page, Western Blot, Fluorescence, Cell Culture, Incubation, Variant Assay, Patch Clamp, Southern Blot, Modification, BAC Assay, Homologous Recombination, Transgenic Assay, Imaging

    Related Articles

    Centrifugation:

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: The beads were removed by centrifugation, and the “precleared” supernatants were added to protein A or G beads that had been preincubated with the primary antibodies or non-immune IgG for 2 h at 4 °C. .. Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C.

    Article Title: Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
    Article Snippet: Blood was collected from a local fish farm and the serum was separated by centrifugation and stored at −20 °C until used. .. N -Glycosidase F (PNGase F) and endoglycosidase buffer pack were purchased from New England Biolabs (Ipswich, MA, USA).

    Amplification:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The full-length membrane-bound form of mouse wild-type Kl cDNA was amplified with Easy A (Agilent Technologies, Stockport, UK) using the forward primer ( 5’- CTC AAG CTT GCT CCC GCA GCA TGC TAG CC -3’ ) and the reverse primer ( 5’- GCAG AAT TCG CTT ATA ACT TCT CTG GCC TTT C -3’ ), and the PCR product sub-cloned into pEGFP-N1 (Clontech, Saint-Germain-en-Laye, France) [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Confocal Microscopy:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Nucleic Acid Electrophoresis:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK). .. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), probed with mouse anti-GFP antibody (Roche Diagnostics, Burgess Hill, UK) followed by HRP-conjugated goat anti-mouse IgG (Bio-Rad, Hemel Hempstead, UK) and visualized by electrochemiluminesence (ECL) detection (GE Healthcare, Little Chalfont, UK), as described [ ].

    Pyrolysis Gas Chromatography:

    Article Title: Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
    Article Snippet: Chemicals and Materials 2,5-Dihydroxybenzoic acid (DHB), dimethyl sulfoxide (DMSO), sodium hydroxide, N-methylmorpholine, acetonitrile (ACN), methylamine hydrochloride, (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP), trifluoroacetic acid (TFA), 1-butanol, ethanol, porous graphitic carbon (PGC), microcrystalline cellulose (MCC) were obtained from Sigma-Aldrich (St. Louis, MO). .. N -Glycosidase F (PNGase F) and endoglycosidase buffer pack were purchased from New England Biolabs (Ipswich, MA, USA).

    Construct:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: Paragraph title: Analysis of the mP2X7-EGFP constructs upon expression in HEK cells ... 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)).

    Incubation:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)). .. For ethidium uptake measurements, cells were seeded after 27 h in 96-well plates (5x104 cells/well) and incubated in the presence of 20 mM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( ).

    Article Title: The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition
    Article Snippet: .. The prepared total cell lysates were diluted with four volumes of endoglycosidase H buffer (62.5 m m sodium citrate, pH 5.5, and 1.25 m m PMSF) and incubated with 20 units/μl endoglycosidase H (Endo Hf ; New England Biolabs, Beverly, MA) at 37 °C for 1 h with occasional mixing by tapping. ..

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: After overnight incubation, the beads were extensively washed with 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, and 0.3% (w/v) Triton X-100. .. Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C.

    Expressing:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: Paragraph title: Analysis of the mP2X7-EGFP constructs upon expression in HEK cells ... 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)).

    Article Title: Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia *
    Article Snippet: .. COS7 cells expressing GlyT2 or the desired mutants were lysed in 1× lysis buffer (150 m m NaCl, 50 m m Tris-HCl (pH 7.4), 5 m m EDTA, 1% Triton X-100, 0.1% SDS, 0.25% deoxycholate sodium, 0.4 m m PMSF, and 4 μ m pepstatin) and digested with the chosen endoglycosidase (peptide: N -glycosidase F (New England Biolabs) or endoglycosidase H or D (Roche Applied Science)) in a small volume of the appropriate buffer, according to the manufacturer's instructions. ..

    Article Title: Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme ▿
    Article Snippet: Regeneration dextrose base (RDB), buffered minimal glycerol (BMG), and buffered minimal methanol (BMM) media were prepared according to the manual of the Pichia Expression kit (Invitrogen, San Diego, CA). .. The AvrII restriction endonuclease and endoglycosidase (Endo Hf ) were purchased from New England Biolabs (Beverly, MA).

    BIA-KA:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: Endoglycosidase (New England Biolabs) treatment was performed for 30 min at 37°C in 20 μl sample aliquots with loading buffer (IUB miliunits: EndoH 10, PNGaseF 20). .. Endoglycosidase (New England Biolabs) treatment was performed for 30 min at 37°C in 20 μl sample aliquots with loading buffer (IUB miliunits: EndoH 10, PNGaseF 20).

    Article Title: A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy
    Article Snippet: Cleared supernatants were treated with endoglycosidase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). .. Cleared supernatants were treated with endoglycosidase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA).

    Modification:

    Article Title: Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia *
    Article Snippet: Paragraph title: Carbohydrate Modification ... COS7 cells expressing GlyT2 or the desired mutants were lysed in 1× lysis buffer (150 m m NaCl, 50 m m Tris-HCl (pH 7.4), 5 m m EDTA, 1% Triton X-100, 0.1% SDS, 0.25% deoxycholate sodium, 0.4 m m PMSF, and 4 μ m pepstatin) and digested with the chosen endoglycosidase (peptide: N -glycosidase F (New England Biolabs) or endoglycosidase H or D (Roche Applied Science)) in a small volume of the appropriate buffer, according to the manufacturer's instructions.

    Article Title: Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)
    Article Snippet: .. Carbohydrate Modification Pulse-chased GlyT2 immunoprecipitates were digested with the desired endoglycosidase (PNGase F, New England Biolabs; or Endoglycosidase H or D, Roche) in a small volume of the appropriate buffer, following the manufacturer’s instructions. .. For tunicamycin treatment, GlyT2-expressing cells were treated with 1–10 µg/ml tunicamycin or the vehicle alone (DMSO) for the time and the temperature indicated in the figure legends, immunoprecipitated with the desired antibodies and resolved by SDS-PAGE.

    Western Blot:

    Article Title: The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition
    Article Snippet: Signal detection was performed using Western Lightning ECL Pro system (PerkinElmer Life Sciences) with a bioimaging analyzer (LAS4000; Fuji Photo Film) or x-ray film. .. The prepared total cell lysates were diluted with four volumes of endoglycosidase H buffer (62.5 m m sodium citrate, pH 5.5, and 1.25 m m PMSF) and incubated with 20 units/μl endoglycosidase H (Endo Hf ; New England Biolabs, Beverly, MA) at 37 °C for 1 h with occasional mixing by tapping.

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: Paragraph title: Immunoprecipitation, Endoglycosidase Digestions, and Western Blotting ... Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C.

    Article Title: A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy
    Article Snippet: GCase trafficking out of the ER was monitored by examining N-glycan carbohydrate processing via Western blotting [ ]. .. Cleared supernatants were treated with endoglycosidase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA).

    Countercurrent Chromatography:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The full-length membrane-bound form of mouse wild-type Kl cDNA was amplified with Easy A (Agilent Technologies, Stockport, UK) using the forward primer ( 5’- CTC AAG CTT GCT CCC GCA GCA TGC TAG CC -3’ ) and the reverse primer ( 5’- GCAG AAT TCG CTT ATA ACT TCT CTG GCC TTT C -3’ ), and the PCR product sub-cloned into pEGFP-N1 (Clontech, Saint-Germain-en-Laye, France) [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Transfection:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: HEK293 cells were cultured and transiently transfected with 1.5-2 mg DNA/well of a 6-well-plate (Lipofectamin, Thermo Fisher Scientific). .. 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)).

    Concentration Assay:

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: .. Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C. .. The reactions were stopped by the addition of Laemmli sample buffer and analyzed by Western blotting as previously described ( ) using anti-rabbit IgG light chains or anti-mouse IgG conjugated to peroxidase (diluted 1:50,000) as secondary antibodies.

    Protease Inhibitor:

    Article Title: A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy
    Article Snippet: Left subcortical tissue was homogenized in 25 mM Bis-Tris, pH 6.5, 150 mM NaCl, 0.1 % Triton-X100 with Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA) using a rotostat. .. Cleared supernatants were treated with endoglycosidase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA).

    Cell Culture:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: HEK293 cells were cultured and transiently transfected with 1.5-2 mg DNA/well of a 6-well-plate (Lipofectamin, Thermo Fisher Scientific). .. 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)).

    other:

    Article Title: Identifying N-linked glycan moiety and motifs in the cysteine-rich domain critical for N-glycosylation and intracellular trafficking of SR-AI and MARCO
    Article Snippet: Peptide N-glycosidase (PNGase F) and endoglycosidase (Endo H) were purchased from New England BioLabs (Ipswich, MA).

    Imaging:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: Protein was either directly visualized by EGFP fluorescence scanning (Typhoon, GE Healthcare) or blotted onto Immobilon-FL PVDF membranes (Merck Millipore) and detected with an Odyssey infrared imaging system (LI-COR Biosciences) using the indicated antibodies (S1 Material and methods). .. Endoglycosidase (New England Biolabs) treatment was performed for 30 min at 37°C in 20 μl sample aliquots with loading buffer (IUB miliunits: EndoH 10, PNGaseF 20).

    Polymerase Chain Reaction:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The full-length membrane-bound form of mouse wild-type Kl cDNA was amplified with Easy A (Agilent Technologies, Stockport, UK) using the forward primer ( 5’- CTC AAG CTT GCT CCC GCA GCA TGC TAG CC -3’ ) and the reverse primer ( 5’- GCAG AAT TCG CTT ATA ACT TCT CTG GCC TTT C -3’ ), and the PCR product sub-cloned into pEGFP-N1 (Clontech, Saint-Germain-en-Laye, France) [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Article Title: Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme ▿
    Article Snippet: Isis proofreading DNA polymerase was from Qbiogene (Carlsbad, CA), and the XhoI restriction endonuclease, T4 DNA ligase, the PCR Clean-Up System (Wizard SV Gel), and the DNA Purification System (Wizard Plus SV Minipreps) were purchased from Promega (Madison, WI). .. The AvrII restriction endonuclease and endoglycosidase (Endo Hf ) were purchased from New England Biolabs (Beverly, MA).

    Recombinant:

    Article Title: Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor *
    Article Snippet: Paragraph title: Production of Recombinant Proteins ... Endoglycosidase (20 μl, Endo Hf 1,000 units/μl, New England Biolabs) were added, and deglycosylation was performed for 24 h at room temperature.

    Immunofluorescence:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    High Performance Liquid Chromatography:

    Article Title: Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis
    Article Snippet: N -glycosidase F (PNGase F) and endoglycosidase buffer pack (EBP) were obtained from New England Biolabs (MA, U.S.A.). .. HPLC grade ACN was from Merck KGaA (Darmstadt, Germany).

    Fluorescence:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)). .. For ethidium uptake measurements, cells were seeded after 27 h in 96-well plates (5x104 cells/well) and incubated in the presence of 20 mM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( ).

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: Protein was either directly visualized by EGFP fluorescence scanning (Typhoon, GE Healthcare) or blotted onto Immobilon-FL PVDF membranes (Merck Millipore) and detected with an Odyssey infrared imaging system (LI-COR Biosciences) using the indicated antibodies (S1 Material and methods). .. Endoglycosidase (New England Biolabs) treatment was performed for 30 min at 37°C in 20 μl sample aliquots with loading buffer (IUB miliunits: EndoH 10, PNGaseF 20).

    Mutagenesis:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Isolation:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: In vitro Studies of Cellular Localisation Total RNA was isolated from kidneys of wild-type mice using the RNeasy mini kit (Qiagen, Crawley, UK) and 2 μg was used to synthesize cDNA using AffinityScript multiple temperature reverse transcriptase (Agilent Technologies, Edinburgh, UK) using methods previously described [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: For endoglycosidase digestions, proteins isolated by immunoprecipitation were eluted from protein A beads by heating at 65 °C in a solution containing 0.5% SDS and 1% β-mercaptoethanol. .. Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C.

    Flow Cytometry:

    Article Title: Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor *
    Article Snippet: During induction, an additional 100 ml of 20% (v/v) methanol was added at a constant flow rate. .. Endoglycosidase (20 μl, Endo Hf 1,000 units/μl, New England Biolabs) were added, and deglycosylation was performed for 24 h at room temperature.

    Mouse Assay:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: In vitro Studies of Cellular Localisation Total RNA was isolated from kidneys of wild-type mice using the RNeasy mini kit (Qiagen, Crawley, UK) and 2 μg was used to synthesize cDNA using AffinityScript multiple temperature reverse transcriptase (Agilent Technologies, Edinburgh, UK) using methods previously described [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Sequencing:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The kl 604N mutation was introduced using site-directed mutagenesis with the forward primer 5’- AC TGG GCC CTG AAC TTG CCT CTG GGT -3’ and its reverse complement, and DNA sequence analysis of the constructs was undertaken, using previously reported methods [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Article Title: Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor *
    Article Snippet: Endoglycosidase (20 μl, Endo Hf 1,000 units/μl, New England Biolabs) were added, and deglycosylation was performed for 24 h at room temperature. .. The purity and identity of the protein were verified by SDS-PAGE and N-terminal sequencing.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy
    Article Snippet: Cleared supernatants were treated with endoglycosidase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). .. The glycoforms were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose.

    Staining:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The wild-type and mutant Kl constructs (1μg of each construct) were transiently transfected into COS-7 cells using jetPEI reagent (Polyplus Transfection, Illkirch, France) and expression visualized by immunofluorescence staining using anti-Golgi matrix protein (GM130) (BD Bioscience, Oxford, UK) or mouse anti-protein disulphide isomerase (PDI) (Enzo Life Science, Exeter, UK) under confocal microscopy, as reported [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Activated Clotting Time Assay:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The full-length membrane-bound form of mouse wild-type Kl cDNA was amplified with Easy A (Agilent Technologies, Stockport, UK) using the forward primer ( 5’- CTC AAG CTT GCT CCC GCA GCA TGC TAG CC -3’ ) and the reverse primer ( 5’- GCAG AAT TCG CTT ATA ACT TCT CTG GCC TTT C -3’ ), and the PCR product sub-cloned into pEGFP-N1 (Clontech, Saint-Germain-en-Laye, France) [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Purification:

    Article Title: Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis
    Article Snippet: N -glycosidase F (PNGase F) and endoglycosidase buffer pack (EBP) were obtained from New England Biolabs (MA, U.S.A.). .. Deionized water was purified using a Milli-Q device (Millipore, MA, U.S.A.).

    Article Title: Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
    Article Snippet: N -Glycosidase F (PNGase F) and endoglycosidase buffer pack were purchased from New England Biolabs (Ipswich, MA, USA). .. All solutions were prepared using deionized water purified by a Milli-Q purification system (Millipore, MA, USA).

    Article Title: Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor *
    Article Snippet: Endoglycosidase (20 μl, Endo Hf 1,000 units/μl, New England Biolabs) were added, and deglycosylation was performed for 24 h at room temperature. .. The solution was diluted in 500 ml of PBS, and Ni-NTA purification was repeated as above.

    SDS Page:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK). .. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), probed with mouse anti-GFP antibody (Roche Diagnostics, Burgess Hill, UK) followed by HRP-conjugated goat anti-mouse IgG (Bio-Rad, Hemel Hempstead, UK) and visualized by electrochemiluminesence (ECL) detection (GE Healthcare, Little Chalfont, UK), as described [ ].

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: .. 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)). .. For ethidium uptake measurements, cells were seeded after 27 h in 96-well plates (5x104 cells/well) and incubated in the presence of 20 mM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( ).

    Article Title: The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition
    Article Snippet: Proteins were separated by SDS-PAGE and transferred to an ImmobilonTM polyvinylidene difluoride membrane (Millipore, Billerica, MA), as described previously ( ). .. The prepared total cell lysates were diluted with four volumes of endoglycosidase H buffer (62.5 m m sodium citrate, pH 5.5, and 1.25 m m PMSF) and incubated with 20 units/μl endoglycosidase H (Endo Hf ; New England Biolabs, Beverly, MA) at 37 °C for 1 h with occasional mixing by tapping.

    Article Title: Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor *
    Article Snippet: Endoglycosidase (20 μl, Endo Hf 1,000 units/μl, New England Biolabs) were added, and deglycosylation was performed for 24 h at room temperature. .. The purity and identity of the protein were verified by SDS-PAGE and N-terminal sequencing.

    Article Title: Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)
    Article Snippet: Carbohydrate Modification Pulse-chased GlyT2 immunoprecipitates were digested with the desired endoglycosidase (PNGase F, New England Biolabs; or Endoglycosidase H or D, Roche) in a small volume of the appropriate buffer, following the manufacturer’s instructions. .. For tunicamycin treatment, GlyT2-expressing cells were treated with 1–10 µg/ml tunicamycin or the vehicle alone (DMSO) for the time and the temperature indicated in the figure legends, immunoprecipitated with the desired antibodies and resolved by SDS-PAGE.

    Software:

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C. .. For quantitative analysis, unsaturated autoradiograms were acquired using an ARCUS II scanner (Agfa-Gevaert, Mortsel, Germany), and the density of each band was quantified using NIH Image J software (National Technical Information Service, Springfield, VA).

    In Vitro:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: Paragraph title: In vitro Studies of Cellular Localisation ... Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Patch Clamp:

    Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
    Article Snippet: 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)). .. Patch-clamp recordings were performed as described ( ) in normal (147 mM NaCl, 2 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM HEPES, and 13 mM glucose) or low divalent cation (0 MgCl2 , 0.1 mM CaCl2 ) containing extracellular solution.

    Immunoprecipitation:

    Article Title: The Regulated Expression, Intracellular Trafficking, and Membrane Recycling of the P2Y-like Receptor GPR17 in Oli-neu Oligodendroglial Cells *
    Article Snippet: Paragraph title: Immunoprecipitation, Endoglycosidase Digestions, and Western Blotting ... Aliquots of each sample were then diluted with Nonidet P-40 (1% final concentration) and 1/10 concentrated endoglycosidase F or endoglycosidase H reaction buffers (G7 or G5, New England Biolabs) in accordance with the manufacturer's instructions and digested with 250 units of endoglycosidase F or H for 1 h at 37 or 0 °C.

    Article Title: Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)
    Article Snippet: Carbohydrate Modification Pulse-chased GlyT2 immunoprecipitates were digested with the desired endoglycosidase (PNGase F, New England Biolabs; or Endoglycosidase H or D, Roche) in a small volume of the appropriate buffer, following the manufacturer’s instructions. .. For tunicamycin treatment, GlyT2-expressing cells were treated with 1–10 µg/ml tunicamycin or the vehicle alone (DMSO) for the time and the temperature indicated in the figure legends, immunoprecipitated with the desired antibodies and resolved by SDS-PAGE.

    DNA Purification:

    Article Title: Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme ▿
    Article Snippet: Isis proofreading DNA polymerase was from Qbiogene (Carlsbad, CA), and the XhoI restriction endonuclease, T4 DNA ligase, the PCR Clean-Up System (Wizard SV Gel), and the DNA Purification System (Wizard Plus SV Minipreps) were purchased from Promega (Madison, WI). .. The AvrII restriction endonuclease and endoglycosidase (Endo Hf ) were purchased from New England Biolabs (Beverly, MA).

    CTG Assay:

    Article Title: N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models
    Article Snippet: The kl 604N mutation was introduced using site-directed mutagenesis with the forward primer 5’- AC TGG GCC CTG AAC TTG CCT CTG GGT -3’ and its reverse complement, and DNA sequence analysis of the constructs was undertaken, using previously reported methods [ ]. .. Briefly, lysates were boiled for 10 min in denaturing buffer (0.5% SDS, 40mM DTT), and treated with endoglycosidase (Endo) H (New England Biolabs, Ipswich, UK).

    Lysis:

    Article Title: Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia *
    Article Snippet: .. COS7 cells expressing GlyT2 or the desired mutants were lysed in 1× lysis buffer (150 m m NaCl, 50 m m Tris-HCl (pH 7.4), 5 m m EDTA, 1% Triton X-100, 0.1% SDS, 0.25% deoxycholate sodium, 0.4 m m PMSF, and 4 μ m pepstatin) and digested with the chosen endoglycosidase (peptide: N -glycosidase F (New England Biolabs) or endoglycosidase H or D (Roche Applied Science)) in a small volume of the appropriate buffer, according to the manufacturer's instructions. ..

    Fluorescence In Situ Hybridization:

    Article Title: Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
    Article Snippet: Blood was collected from a local fish farm and the serum was separated by centrifugation and stored at −20 °C until used. .. N -Glycosidase F (PNGase F) and endoglycosidase buffer pack were purchased from New England Biolabs (Ipswich, MA, USA).

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    New England Biolabs endoglycosidase h reaction buffers
    Endoglycosidase H Reaction Buffers, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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