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Roche netn buffer
Netn Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 4 article reviews
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netn buffer - by Bioz Stars, 2020-04
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Clone Assay:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: AIMP2 gene was amplified by PCR in order to insert SalΙ/BamHΙ sites and cloned into the pIRES2-EGFP-SBP. .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Centrifugation:

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics). .. After 20 minutes of lysis, protein lysates were cleared by centrifugation at 12,000 rpm at 4°C for 10 minutes.

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics). .. Cell debris was pelleted by centrifugation (12,000 rpm, 10 min, 4 °C) and the supernatant was collected.

Article Title: Metformin pharmacogenomics: a genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines
Article Snippet: .. As described previously , cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet p-40) containing protease and phosphatase inhibitors (Roche) on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4ºC, cell lysate were incubated with 4 µg of antibody or normal IgG (Cell Signalling Technology) and protein A Sepharose beads (Amersham Biosciences) overnight at 4ºC. ..

Amplification:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: AIMP2 gene was amplified by PCR in order to insert SalΙ/BamHΙ sites and cloned into the pIRES2-EGFP-SBP. .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Autoradiography:

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ). .. Radiolabelled proteins in dried gels were visualized by autoradiography.

Incubation:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: .. For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation. .. For streptavidin-agarose pulldowns, beads were incubated in 50 µL 1× phosphate-buffered saline (PBS) containing either 10 µM free biotin, 10 µM PAR-biotin (Trevigen) or 10 µM H3K9me3-biotin (Epicypher) for 0.5 h at 4 °C, with rotation, before being quenched by incubation with 500 µL of 1 mM TRIS pH 8.8 containing 80 µM free biotin for 15 min at 4 °C, with rotation, and washed 2× with 1 mL NETN buffer.

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: Transfected HEK293 cells on 6-well plates were incubated with 1 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min at 4 °C. .. After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche).

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase
Article Snippet: .. Pellets were resuspended in NETN buffer with protease inhibitors (Roche), sonicated, cleared, and incubated with glutathione-Sepharose beads (Amersham Biosciences). .. The beads were washed, and GST-DCNL1 was eluted using GST elution buffer (30 mM glutathione, 50 mM Tris, pH 8). pET-46 EK/LIC VHL(1–155) and pET-15b-DCNL1 were expressed in Rosetta-2 and BL21 cells, respectively, and induced with IPTG.

Article Title: Facioscapulohumeral muscular dystrophy region gene 1 (FRG1) is a dynamic RNA-associated and actin bundling protein
Article Snippet: Bacterial cells were dissolved in NETN buffer [20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, protease inhibitor cocktail (Roche)] 3 hrs after induction and lysed by French press (American Instrument Company, Haverhill, MA, USA). .. These soluble fractions (100 µl) were incubated with 40 µl of a 75% glutathione bead slurry (4B, Amersham Pharmacia Biotech, Buckinghamshire, UK), rotating for 1 hr at 4°C.

Article Title: Metformin pharmacogenomics: a genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines
Article Snippet: .. As described previously , cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet p-40) containing protease and phosphatase inhibitors (Roche) on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4ºC, cell lysate were incubated with 4 µg of antibody or normal IgG (Cell Signalling Technology) and protein A Sepharose beads (Amersham Biosciences) overnight at 4ºC. ..

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: .. Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ). ..

Expressing:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics). .. Then, protein amount was measured by Bradford assay and the protein expression was confirmed by immunoblotting.

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: The pcDNA3.1 BCL11A-XL and associated mutant expression vectors were in vitro transcribed/translated in the presence of [35 S]-methionine in reticulocyte lysate (Promega) according to the manufacturer’s instructions. .. Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ).

Bradford Assay:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics). .. Then, protein amount was measured by Bradford assay and the protein expression was confirmed by immunoblotting.

Modification:

Article Title: De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair
Article Snippet: Chromatin fractionation Cells were first lysed with NETN buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT) plus protease and phosphatase inhibitor cocktails (Roche). .. The fractions were lysed again with the modified NETN buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 50 U/mL MNase, 50 U/ml Benzonase) plus protease and phosphatase inhibitor cocktails (Roche).

Article Title: The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response
Article Snippet: Cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and antibiotics. .. Cells were lysed with RIPA or NETN buffer containing protease and phosphatase inhibitors (Roche).

Western Blot:

Article Title: De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair
Article Snippet: Chromatin fractionation Cells were first lysed with NETN buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT) plus protease and phosphatase inhibitor cocktails (Roche). .. Finally, the soluble fractions were used for western blots.

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: Paragraph title: Western Blotting ... Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics).

Article Title: The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response
Article Snippet: Paragraph title: Cell culture, cell lysis, immunoprecipitation, and western blotting ... Cells were lysed with RIPA or NETN buffer containing protease and phosphatase inhibitors (Roche).

Article Title: The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2–mediated axon guidance
Article Snippet: .. Western blot analysis and reagents Cell lysates were prepared in NETN buffer containing 1 mmol/liter NaF, 2.5 mmol/liter β-glycerophosphate, and a protease inhibitor cocktail (Roche). ..

Transfection:

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: Transfected HEK293 cells on 6-well plates were incubated with 1 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min at 4 °C. .. After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche).

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: Paragraph title: AIMP1, AIMP2 and KARS Transfection ... After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Protease Inhibitor:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: .. For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation. .. For streptavidin-agarose pulldowns, beads were incubated in 50 µL 1× phosphate-buffered saline (PBS) containing either 10 µM free biotin, 10 µM PAR-biotin (Trevigen) or 10 µM H3K9me3-biotin (Epicypher) for 0.5 h at 4 °C, with rotation, before being quenched by incubation with 500 µL of 1 mM TRIS pH 8.8 containing 80 µM free biotin for 15 min at 4 °C, with rotation, and washed 2× with 1 mL NETN buffer.

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: .. After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche). .. Biotinylated surface proteins were purified using NeutrAvidin-agarose (Pierce), washed, and resuspended in protein loading buffer.

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: .. Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics). .. After 20 minutes of lysis, protein lysates were cleared by centrifugation at 12,000 rpm at 4°C for 10 minutes.

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics). .. Cell debris was pelleted by centrifugation (12,000 rpm, 10 min, 4 °C) and the supernatant was collected.

Article Title: Facioscapulohumeral muscular dystrophy region gene 1 (FRG1) is a dynamic RNA-associated and actin bundling protein
Article Snippet: .. Bacterial cells were dissolved in NETN buffer [20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, protease inhibitor cocktail (Roche)] 3 hrs after induction and lysed by French press (American Instrument Company, Haverhill, MA, USA). ..

Article Title: The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets ▿ Visual System by Regulating Specific RNA Targets ▿ †
Article Snippet: .. Anterior quarters of 600 third-instar larvae were dissected in cold 1× phosphate-buffered saline and pooled in 1 ml of cold NETN buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5 mM MgCl2, 0.25% NP-40, 0.5% Triton X-100) with 400 μg tRNA from Escherichia coli (Roche), 20 μl RNasin (Promega), and 100 μl protease inhibitor cocktail (complete, EDTA-free; Roche). .. Tissue extracts were homogenized with a plastic pestle in a 1.5-ml microcentrifuge tube and sonicated twice for 5 seconds.

Article Title: The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2–mediated axon guidance
Article Snippet: .. Western blot analysis and reagents Cell lysates were prepared in NETN buffer containing 1 mmol/liter NaF, 2.5 mmol/liter β-glycerophosphate, and a protease inhibitor cocktail (Roche). ..

Cell Culture:

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: .. Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics). .. After 20 minutes of lysis, protein lysates were cleared by centrifugation at 12,000 rpm at 4°C for 10 minutes.

Article Title: The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response
Article Snippet: Paragraph title: Cell culture, cell lysis, immunoprecipitation, and western blotting ... Cells were lysed with RIPA or NETN buffer containing protease and phosphatase inhibitors (Roche).

Polymerase Chain Reaction:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: AIMP2 gene was amplified by PCR in order to insert SalΙ/BamHΙ sites and cloned into the pIRES2-EGFP-SBP. .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Sonication:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: .. For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation. .. For streptavidin-agarose pulldowns, beads were incubated in 50 µL 1× phosphate-buffered saline (PBS) containing either 10 µM free biotin, 10 µM PAR-biotin (Trevigen) or 10 µM H3K9me3-biotin (Epicypher) for 0.5 h at 4 °C, with rotation, before being quenched by incubation with 500 µL of 1 mM TRIS pH 8.8 containing 80 µM free biotin for 15 min at 4 °C, with rotation, and washed 2× with 1 mL NETN buffer.

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase
Article Snippet: .. Pellets were resuspended in NETN buffer with protease inhibitors (Roche), sonicated, cleared, and incubated with glutathione-Sepharose beads (Amersham Biosciences). .. The beads were washed, and GST-DCNL1 was eluted using GST elution buffer (30 mM glutathione, 50 mM Tris, pH 8). pET-46 EK/LIC VHL(1–155) and pET-15b-DCNL1 were expressed in Rosetta-2 and BL21 cells, respectively, and induced with IPTG.

Article Title: The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets ▿ Visual System by Regulating Specific RNA Targets ▿ †
Article Snippet: Anterior quarters of 600 third-instar larvae were dissected in cold 1× phosphate-buffered saline and pooled in 1 ml of cold NETN buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5 mM MgCl2, 0.25% NP-40, 0.5% Triton X-100) with 400 μg tRNA from Escherichia coli (Roche), 20 μl RNasin (Promega), and 100 μl protease inhibitor cocktail (complete, EDTA-free; Roche). .. Tissue extracts were homogenized with a plastic pestle in a 1.5-ml microcentrifuge tube and sonicated twice for 5 seconds.

Recombinant:

Article Title: Facioscapulohumeral muscular dystrophy region gene 1 (FRG1) is a dynamic RNA-associated and actin bundling protein
Article Snippet: To test the FRG1-KPNA2 interaction, recombinant GST protein inductions (50 ml) were performed in E. coli DH5α bacterial cells using 1mM IPTG. .. Bacterial cells were dissolved in NETN buffer [20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, protease inhibitor cocktail (Roche)] 3 hrs after induction and lysed by French press (American Instrument Company, Haverhill, MA, USA).

Nucleic Acid Electrophoresis:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation. .. For IB, washed IPs or 25-50 µg lysates (inputs) were incubated at 95 °C for 5 min in 1× Laemmli SDS Sample buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Mutagenesis:

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: The pcDNA3.1 BCL11A-XL and associated mutant expression vectors were in vitro transcribed/translated in the presence of [35 S]-methionine in reticulocyte lysate (Promega) according to the manufacturer’s instructions. .. Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ).

Isolation:

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: Biotinylation assay HEK293 cells were biotinylated using the Pierce Cell Surface Protein Isolation Kit (Pierce, 89881). .. After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche).

Purification:

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche). .. Biotinylated surface proteins were purified using NeutrAvidin-agarose (Pierce), washed, and resuspended in protein loading buffer.

Article Title: Facioscapulohumeral muscular dystrophy region gene 1 (FRG1) is a dynamic RNA-associated and actin bundling protein
Article Snippet: Bacterial cells were dissolved in NETN buffer [20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, protease inhibitor cocktail (Roche)] 3 hrs after induction and lysed by French press (American Instrument Company, Haverhill, MA, USA). .. To test the FRG1-TAP interaction, recombinant GST and GST-Tap proteins were purified and bound to glutathione beads as described .

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: GST pull-down assays GST fusion proteins were expressed in Escherichia coli BL21 using isopropyl-β-d -thiogalactopyranoside induction, and purified on glutathione-Sepharose beads (Amersham Biosciences). .. Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ).

Protein Purification:

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase
Article Snippet: Paragraph title: Protein purification. ... Pellets were resuspended in NETN buffer with protease inhibitors (Roche), sonicated, cleared, and incubated with glutathione-Sepharose beads (Amersham Biosciences).

Protein Extraction:

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase
Article Snippet: Pellets were resuspended in NETN buffer with protease inhibitors (Roche), sonicated, cleared, and incubated with glutathione-Sepharose beads (Amersham Biosciences). .. Pellets were resuspended in Ni-nitrilotriacetic acid (NTA) buffer (50 mM Tris, pH 8, 300 mM NaCl, 20 mM imidazole, 1× bacterial protein extraction reagent [B-PER]) with protease inhibitors (Roche), sonicated, cleared, and incubated with Ni-NTA (Invitrogen).

SDS Page:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation. .. For IB, washed IPs or 25-50 µg lysates (inputs) were incubated at 95 °C for 5 min in 1× Laemmli SDS Sample buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics). .. The proteins were fractionated by SDS-PAGE, electro-transferred to Immobilon-P membranes (EMD Millipore), blotted with the indicated primary antibodies followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and detected using SuperSignal West Pico or Femto Chemiluminescence Substrate (Thermo Fisher Scientific).

Article Title: Metformin pharmacogenomics: a genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines
Article Snippet: As described previously , cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet p-40) containing protease and phosphatase inhibitors (Roche) on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4ºC, cell lysate were incubated with 4 µg of antibody or normal IgG (Cell Signalling Technology) and protein A Sepharose beads (Amersham Biosciences) overnight at 4ºC. .. The precipitates were then eluted in SDS sample buffer and separated by SDS-PAGE.

Article Title: The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2–mediated axon guidance
Article Snippet: Western blot analysis and reagents Cell lysates were prepared in NETN buffer containing 1 mmol/liter NaF, 2.5 mmol/liter β-glycerophosphate, and a protease inhibitor cocktail (Roche). .. Cell lysates were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane.

Plasmid Preparation:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: As a negative control, a mock vector having only the S/FLAG/SBP tag was also transfected into the cell lines. .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Negative Control:

Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Article Snippet: As a negative control, a mock vector having only the S/FLAG/SBP tag was also transfected into the cell lines. .. After incubating 30 h, cells were harvested at a confluence of 90~100% and lysed by NETN buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, Protease Inhibitor Cocktail (Roche Diagnostics) and Phosphatase Inhibitor Cocktail (Roche Diagnostics).

Positron Emission Tomography:

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase
Article Snippet: Pellets were resuspended in NETN buffer with protease inhibitors (Roche), sonicated, cleared, and incubated with glutathione-Sepharose beads (Amersham Biosciences). .. The beads were washed, and GST-DCNL1 was eluted using GST elution buffer (30 mM glutathione, 50 mM Tris, pH 8). pET-46 EK/LIC VHL(1–155) and pET-15b-DCNL1 were expressed in Rosetta-2 and BL21 cells, respectively, and induced with IPTG.

In Vitro:

Article Title: A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors
Article Snippet: The pcDNA3.1 BCL11A-XL and associated mutant expression vectors were in vitro transcribed/translated in the presence of [35 S]-methionine in reticulocyte lysate (Promega) according to the manufacturer’s instructions. .. Equalised amounts of GST proteins were incubated with 35 S-radiolabelled protein in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) containing 1× complete protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10−6 M cognate ligand as described previously ( ).

Cell Surface Biotinylation Assay:

Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
Article Snippet: Paragraph title: Biotinylation assay ... After quenching the biotinylation reaction with 50 mM glycine and rinsing of the cells with ice-cold TBS (Tris-buffered saline) and PBS, cells were scrapped from the plates and lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris/HCl, pH 7.4) supplemented with an EDTA-free protease inhibitor mixture (Roche).

Immunoprecipitation:

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor
Article Snippet: Paragraph title: Immunoblotting (IB) and immunoprecipitation (IP) ... For IB or IP, cells were lysed in 5× packed cell volume of ice-cold NETN buffer (150 mM NaCl, 0.2 mM EDTA, 50 mM Tris-HCl pH 7.5 and 1% (v/v) NP-40 detergent) supplemented with 1× protease inhibitor cocktail (Roche), 1 mM DTT, PMSF, 1 µM WM, 10 mM NEM, 5 µM PARGi, 3-AB, and 0.1 µM MC-LR and sonicated (10%, 5 s) and clarified at 10,000 rpm for 10 min. For GFP IP examining interactions with PAR, 2 mg of lysate was incubated with 5 µL packed, equilibrated GFP Trap beads (Chromotek) for 2 h at 4 °C with rotation.

Article Title: Metformin pharmacogenomics: a genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines
Article Snippet: Paragraph title: Immunoprecipitation and immunoblotting ... As described previously , cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet p-40) containing protease and phosphatase inhibitors (Roche) on ice for 30 min. After centrifugation at 14,000 g for 10 min at 4ºC, cell lysate were incubated with 4 µg of antibody or normal IgG (Cell Signalling Technology) and protein A Sepharose beads (Amersham Biosciences) overnight at 4ºC.

Article Title: The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets ▿ Visual System by Regulating Specific RNA Targets ▿ †
Article Snippet: Paragraph title: Larval extract preparation and immunoprecipitation. ... Anterior quarters of 600 third-instar larvae were dissected in cold 1× phosphate-buffered saline and pooled in 1 ml of cold NETN buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5 mM MgCl2, 0.25% NP-40, 0.5% Triton X-100) with 400 μg tRNA from Escherichia coli (Roche), 20 μl RNasin (Promega), and 100 μl protease inhibitor cocktail (complete, EDTA-free; Roche).

Article Title: The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response
Article Snippet: Paragraph title: Cell culture, cell lysis, immunoprecipitation, and western blotting ... Cells were lysed with RIPA or NETN buffer containing protease and phosphatase inhibitors (Roche).

Fractionation:

Article Title: De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair
Article Snippet: .. Chromatin fractionation Cells were first lysed with NETN buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT) plus protease and phosphatase inhibitor cocktails (Roche). ..

Lysis:

Article Title: Food Restriction Ameliorates the Development of Polycystic Kidney Disease
Article Snippet: Mouse kidney tissue and cultured cells were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with 5 mM NaF, 50 mM 2-glycerophosphate, and a protease inhibitor cocktail (Roche Diagnostics). .. After 20 minutes of lysis, protein lysates were cleared by centrifugation at 12,000 rpm at 4°C for 10 minutes.

Article Title: The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response
Article Snippet: Paragraph title: Cell culture, cell lysis, immunoprecipitation, and western blotting ... Cells were lysed with RIPA or NETN buffer containing protease and phosphatase inhibitors (Roche).

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  • 94
    Roche netn lysis buffer
    SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to <t>immunoprecipitation</t> (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with <t>NETN</t> lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.
    Netn Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/netn lysis buffer/product/Roche
    Average 94 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    netn lysis buffer - by Bioz Stars, 2020-04
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    92
    Roche netn buffer
    SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to <t>immunoprecipitation</t> (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with <t>NETN</t> lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.
    Netn Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/netn buffer/product/Roche
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    netn buffer - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    Image Search Results


    SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to immunoprecipitation (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with NETN lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.

    Journal: Nucleic Acids Research

    Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

    doi: 10.1093/nar/gkz769

    Figure Lengend Snippet: SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to immunoprecipitation (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with NETN lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.

    Article Snippet: Cell lysis and immunoprecipitation Cells were lysed with ice-cold NETN lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM sodium chloride [NaCl], 0.4% NP-40, 1 mM EDTA), and protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice.

    Techniques: Filtration, Immunoprecipitation, Lysis, Staining, Double Knockout, Knock-Out