binding buffer  (Millipore)


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    Structured Review

    Millipore binding buffer
    Binding Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/binding buffer/product/Millipore
    Average 99 stars, based on 1061 article reviews
    Price from $9.99 to $1999.99
    binding buffer - by Bioz Stars, 2020-04
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    Centrifugation:

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA). .. After being resuspended, cells were lysed via sonication and then debris was removed by centrifugation for 30 min at 11,000 g. The protein in the supernatant was purified using amylose affinity chromatography (New England BioLabs, Ipswich, MA), and then concentrated using Amicon Ultra-15 filters (Millipore, Schwalbach, Germany).

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C. .. The supernatant was applied to Ni-NTA His-binding resin (Novagen), which was pre-equilibrated with the binding buffer containing 6 M urea.

    Filtration:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: The eluate was then loaded on a Superose 6HR (Pharmacia) gel filtration column at 350 mM KCl. .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Stable Transfection:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: At 48 h posttransfection (no stably integrated cell lines could be established for these two proteins), nuclear extracts were prepared, adjusted to 150 mM NaCl, and immunoprecipitated as follows. .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Cytometry:

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: To test binding ability of NPC2 proteins to different bacterial components, plate ELISA assays were performed. .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature.

    Incubation:

    Article Title: Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion
    Article Snippet: .. Attached cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at 1 × 106 cells/ml and incubated with APC-conjugated Annexin V (5 μl /100 μl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 μl of binding buffer containing 1 μl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed using Accuri C6 and CFlow software (Becton Dickinson).

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: Nuclear extracts prepared from FLAG-ING3- or FLAG-MRG15-expressing cells were precleared with protein A-Sepharose (Amersham Bioscience) for 45 min and then incubated with α-FLAG M2 resin (Sigma) overnight at 4°C. .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Article Title: dSLo Interacting Protein 1, a Novel Protein That Interacts with Large-Conductance Calcium-Activated Potassium Channels
    Article Snippet: .. Of the glutathione–agarose beads, 15 μl was combined with solubilized His–tag fusion proteins (∼100 μg) into 1 ml of binding buffer [ containing (in m m ) 10 HEPES, pH 7.5, 0.5 DTT, 0.5 EDTA, 150 NaCl, and 0.2 PMSF with 0.1% NP-40 and 5 mg/ml BSA], incubated for 12 hr at 4°C, and washed three to five times with excess binding buffer; bound proteins were batch-eluted with 30 μl of reduced glutathione (Sigma). .. Eluted proteins (10 μl) were mixed with 2× loading buffer and subjected to 8% SDS-PAGE.

    Article Title: An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides
    Article Snippet: .. Wells were washed three times with binding buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) to remove unbound receptor and 50 μL of 2 μM biotinylated peptide in binding buffer were added and allowed to incubate for 1 h. The wells were washed four times with binding buffer and then incubated with 50 μL of 1 μg/mL streptavidin conjugated with peroxidase (Sigma-Aldrich) for 1 h. The wells were washed five times with binding buffer and 50 μL of peroxidase substrate (1-Step™ Ultra TMB, Pierce) were added. ..

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below. .. Beads were washed three times with binding buffer, followed by immunoblot analysis as described before.

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature. ..

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    Activity Assay:

    Article Title: An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides
    Article Snippet: Wells were washed three times with binding buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) to remove unbound receptor and 50 μL of 2 μM biotinylated peptide in binding buffer were added and allowed to incubate for 1 h. The wells were washed four times with binding buffer and then incubated with 50 μL of 1 μg/mL streptavidin conjugated with peroxidase (Sigma-Aldrich) for 1 h. The wells were washed five times with binding buffer and 50 μL of peroxidase substrate (1-Step™ Ultra TMB, Pierce) were added. .. Bound streptavidin was quantitated by the specific activity of peroxidase (absorbance/ng protein conjugate/min) under the conditions of the assay.

    Expressing:

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA).

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: Protein expression was induced by adding 0·4 mM isopropyl β-D-thiogalactoside (IPTG; Duchefa, Zwijndrecht, the Netherlands) for 4 h at 37°C. .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C.

    Western Blot:

    Article Title: dSLo Interacting Protein 1, a Novel Protein That Interacts with Large-Conductance Calcium-Activated Potassium Channels
    Article Snippet: Of the glutathione–agarose beads, 15 μl was combined with solubilized His–tag fusion proteins (∼100 μg) into 1 ml of binding buffer [ containing (in m m ) 10 HEPES, pH 7.5, 0.5 DTT, 0.5 EDTA, 150 NaCl, and 0.2 PMSF with 0.1% NP-40 and 5 mg/ml BSA], incubated for 12 hr at 4°C, and washed three to five times with excess binding buffer; bound proteins were batch-eluted with 30 μl of reduced glutathione (Sigma). .. The gel was prepared as a Western blot and probed with either dSlo or dSLIP1 antiserum, as described above.

    Kinase Assay:

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below. .. Beads were washed three times with binding buffer, followed by immunoblot analysis as described before.

    Transformation Assay:

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: For the expression and purification of CEA fusion proteins, E. coli BL21 (DE3) strains (Novagen) were transformed with either pET-CEA or pET-Tat-CEA. .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C.

    Transfection:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: Paragraph title: Transfections and immunoprecipitations. ... The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Ligand Binding Assay:

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: Paragraph title: 2.5 Ligand binding assay ... Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature.

    Northern Blot:

    Article Title: dSLo Interacting Protein 1, a Novel Protein That Interacts with Large-Conductance Calcium-Activated Potassium Channels
    Article Snippet: Drosophila embryos (∼1 gm; 0–24 hr) were homogenized with a Dounce homogenizer, and poly(A+ ) mRNA was oligo-dT-selected; 3 μg was prepared as a Northern blot and probed with radiolabeled full-length dSLIP1 antisense RNA (50% formamide, 5% SDS, 400 m m NaPO4 , pH 7.2, and 1 m m EDTA at 65°C overnight and then washed in 1% SDS and 0.5× SSC at 50°C). .. Of the glutathione–agarose beads, 15 μl was combined with solubilized His–tag fusion proteins (∼100 μg) into 1 ml of binding buffer [ containing (in m m ) 10 HEPES, pH 7.5, 0.5 DTT, 0.5 EDTA, 150 NaCl, and 0.2 PMSF with 0.1% NP-40 and 5 mg/ml BSA], incubated for 12 hr at 4°C, and washed three to five times with excess binding buffer; bound proteins were batch-eluted with 30 μl of reduced glutathione (Sigma).

    Infection:

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Sf9 cells at a density of 2-10 6 cells/ml were infected with the recombinant baculoviruses and the recombinant proteins were harvested at 72 h after infection. .. Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA).

    Sonication:

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA). .. After being resuspended, cells were lysed via sonication and then debris was removed by centrifugation for 30 min at 11,000 g. The protein in the supernatant was purified using amylose affinity chromatography (New England BioLabs, Ipswich, MA), and then concentrated using Amicon Ultra-15 filters (Millipore, Schwalbach, Germany).

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C. .. The supernatant was applied to Ni-NTA His-binding resin (Novagen), which was pre-equilibrated with the binding buffer containing 6 M urea.

    Recombinant:

    Article Title: An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides
    Article Snippet: His-tagged or Fc-fusion recombinant receptors (R & D Systems, Minneapolis, MN) were reconstituted in PBS. .. Wells were washed three times with binding buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) to remove unbound receptor and 50 μL of 2 μM biotinylated peptide in binding buffer were added and allowed to incubate for 1 h. The wells were washed four times with binding buffer and then incubated with 50 μL of 1 μg/mL streptavidin conjugated with peroxidase (Sigma-Aldrich) for 1 h. The wells were washed five times with binding buffer and 50 μL of peroxidase substrate (1-Step™ Ultra TMB, Pierce) were added.

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA).

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below. .. Beads were washed three times with binding buffer, followed by immunoblot analysis as described before.

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature. ..

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    Mutagenesis:

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: The following ligands were used: smooth lipopolysaccharide (LPS) from Salmonella enteric , E. coli 055:B5, E. coli 0111:B4; rough mutants of LPS from E. coli EH100 (Ra mutant), E. coli F583 (Rd mutant), E. coli J5 (Rc mutant), and S. enterica serotype minnesota Re 595 (Re mutant), lipid A monophosphoryl from E. coli F583 (Rd mutant), lipid A diphosphoryl from E. coli F583 (Rd mutant) (all from Sigma-Aldrich); TLRgrade lipoteichoic acid (LTA) and peptidoglycan (PG) from B. subtilis and S. aureus , TLRgrade LPS and PG from E. coli K12 (all from Invivogen). .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature.

    Flow Cytometry:

    Article Title: A Novel, Single, Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel Protein 1
    Article Snippet: .. Testis membrane protein (∼1 mg) was solubilized with 1% Triton X-100 in 250 μl of binding buffer (50 mM Na2 HPO4 , 300 mM NaCl, and 1× protease inhibitor cocktail [pH 8.0]) at 4°C for 1 h. Soluble protein was added to 100 μl of buffer-equilibrated cobalt resin (Clontech) supplemented with 20 mM imidazole (pH 7.5) in 250 μl of binding buffer and mixed at 4°C for 2 h. Following a quick spinning of the protein-resin mixture, the supernatant (flow through) was collected and concentrated with a Microcon-50 ultrafiltration column (Millipore, Bedford, MA), and the cobalt resin was washed twice with binding buffer. .. Bound protein was eluted with 300 mM imidazole in binding buffer.

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    Purification:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch. .. For the TAP purification of transiently transfected MRG15-TAP, nuclear extracts were incubated with IgG-Sepharose beads (Amersham Bioscience) overnight at 4°C.

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA).

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below. .. Beads were washed three times with binding buffer, followed by immunoblot analysis as described before.

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature. ..

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: Paragraph title: Purification of CEA fusion proteins ... Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C.

    LDH Cytotoxicity Assay:

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    SDS Page:

    Article Title: dSLo Interacting Protein 1, a Novel Protein That Interacts with Large-Conductance Calcium-Activated Potassium Channels
    Article Snippet: Of the glutathione–agarose beads, 15 μl was combined with solubilized His–tag fusion proteins (∼100 μg) into 1 ml of binding buffer [ containing (in m m ) 10 HEPES, pH 7.5, 0.5 DTT, 0.5 EDTA, 150 NaCl, and 0.2 PMSF with 0.1% NP-40 and 5 mg/ml BSA], incubated for 12 hr at 4°C, and washed three to five times with excess binding buffer; bound proteins were batch-eluted with 30 μl of reduced glutathione (Sigma). .. Eluted proteins (10 μl) were mixed with 2× loading buffer and subjected to 8% SDS-PAGE.

    Plasmid Preparation:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: 293T cells were transfected with 15 μg of either pcDNA3-FLAG-ING3, pcDNA3-TAP-MRG15, or the empty pcDNA3 vector by the calcium phosphate method. .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Software:

    Article Title: Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion
    Article Snippet: Attached cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at 1 × 106 cells/ml and incubated with APC-conjugated Annexin V (5 μl /100 μl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 μl of binding buffer containing 1 μl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed using Accuri C6 and CFlow software (Becton Dickinson).

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    Electrophoresis:

    Article Title: A Novel, Single, Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel Protein 1
    Article Snippet: Testis membrane protein (∼1 mg) was solubilized with 1% Triton X-100 in 250 μl of binding buffer (50 mM Na2 HPO4 , 300 mM NaCl, and 1× protease inhibitor cocktail [pH 8.0]) at 4°C for 1 h. Soluble protein was added to 100 μl of buffer-equilibrated cobalt resin (Clontech) supplemented with 20 mM imidazole (pH 7.5) in 250 μl of binding buffer and mixed at 4°C for 2 h. Following a quick spinning of the protein-resin mixture, the supernatant (flow through) was collected and concentrated with a Microcon-50 ultrafiltration column (Millipore, Bedford, MA), and the cobalt resin was washed twice with binding buffer. .. Flow through and eluate were mixed with LDS loading buffer and heated for 15 min at 70°C before electrophoresis.

    Binding Assay:

    Article Title: Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion
    Article Snippet: .. Attached cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at 1 × 106 cells/ml and incubated with APC-conjugated Annexin V (5 μl /100 μl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 μl of binding buffer containing 1 μl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed using Accuri C6 and CFlow software (Becton Dickinson).

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch. .. For the TAP purification of transiently transfected MRG15-TAP, nuclear extracts were incubated with IgG-Sepharose beads (Amersham Bioscience) overnight at 4°C.

    Article Title: dSLo Interacting Protein 1, a Novel Protein That Interacts with Large-Conductance Calcium-Activated Potassium Channels
    Article Snippet: .. Of the glutathione–agarose beads, 15 μl was combined with solubilized His–tag fusion proteins (∼100 μg) into 1 ml of binding buffer [ containing (in m m ) 10 HEPES, pH 7.5, 0.5 DTT, 0.5 EDTA, 150 NaCl, and 0.2 PMSF with 0.1% NP-40 and 5 mg/ml BSA], incubated for 12 hr at 4°C, and washed three to five times with excess binding buffer; bound proteins were batch-eluted with 30 μl of reduced glutathione (Sigma). .. Eluted proteins (10 μl) were mixed with 2× loading buffer and subjected to 8% SDS-PAGE.

    Article Title: An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides
    Article Snippet: .. Wells were washed three times with binding buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) to remove unbound receptor and 50 μL of 2 μM biotinylated peptide in binding buffer were added and allowed to incubate for 1 h. The wells were washed four times with binding buffer and then incubated with 50 μL of 1 μg/mL streptavidin conjugated with peroxidase (Sigma-Aldrich) for 1 h. The wells were washed five times with binding buffer and 50 μL of peroxidase substrate (1-Step™ Ultra TMB, Pierce) were added. ..

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: .. Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA). .. After being resuspended, cells were lysed via sonication and then debris was removed by centrifugation for 30 min at 11,000 g. The protein in the supernatant was purified using amylose affinity chromatography (New England BioLabs, Ipswich, MA), and then concentrated using Amicon Ultra-15 filters (Millipore, Schwalbach, Germany).

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: .. Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below. .. Beads were washed three times with binding buffer, followed by immunoblot analysis as described before.

    Article Title: Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways
    Article Snippet: .. Purified recombinant NPC2a, NPC2e, NPC2h fusion proteins and the control TRX protein were diluted in binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/ml BSA to 100 nM and added to each well of the coated plates (50 µl/well) and incubated at room temperature for 3 h. Plates were washed with binding buffer four times (each for 5 min), and mouse monoclonal anti-polyHistidine antibody (Sigma, 1:3000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for 2 h. Then plates were washed again, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, 1:2000 dilution in binding buffer containing 0.1 mg/ml BSA) was added (100 µl/well) and incubated at 37°C for another 2 h. At last, p -nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine, 0.5 mM MgCl2 ) was added (50 µl/well) to the plates and incubated for 20 min at room temperature. ..

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C. .. The supernatant was applied to Ni-NTA His-binding resin (Novagen), which was pre-equilibrated with the binding buffer containing 6 M urea.

    Article Title: A Novel, Single, Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel Protein 1
    Article Snippet: .. Testis membrane protein (∼1 mg) was solubilized with 1% Triton X-100 in 250 μl of binding buffer (50 mM Na2 HPO4 , 300 mM NaCl, and 1× protease inhibitor cocktail [pH 8.0]) at 4°C for 1 h. Soluble protein was added to 100 μl of buffer-equilibrated cobalt resin (Clontech) supplemented with 20 mM imidazole (pH 7.5) in 250 μl of binding buffer and mixed at 4°C for 2 h. Following a quick spinning of the protein-resin mixture, the supernatant (flow through) was collected and concentrated with a Microcon-50 ultrafiltration column (Millipore, Bedford, MA), and the cobalt resin was washed twice with binding buffer. .. Bound protein was eluted with 300 mM imidazole in binding buffer.

    Article Title: Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters
    Article Snippet: .. LDH release was measured using the LDH-Cytotoxicity Assay kit (Abcam, AB65391) according to the manufacturer’s instructions, For flow cytometry, cells were trypsinized, spun at 200 g for 10 min, washed with ice-cold PBS, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) at 1 × 106 cells/ml and incubated with (APC)-conjugated recombinant human ANXA5 (5 µl/100 µl cell suspension, BD PharMingen, 550475) at room temperature for 15 min. 400 µl of binding buffer containing 1 µl propidium iodide (1 mg/ml, Sigma-Aldrich, P4864) was then added and incubated on ice for 5 min. .. Cells were analyzed with a C6 Flow cytometer and CFlow software (Becton Dickinson).

    In Vitro:

    Article Title: Cyclin-dependent kinase 1 (CDK1) and CDK2 have opposing roles in regulating interactions of splicing factor 3B1 with chromatin
    Article Snippet: Paragraph title: In vitro binding assay ... Next, beads were washed once with binding buffer (0.1% Triton X-100, 1.2 m m EDTA, 1.67 m m Tris-HCl, pH 8.0, 100 m m NaCl) and resuspended in 1 ml of binding buffer, followed by incubation with 1 μg of purified histone H1 (Sigma-Aldrich, calf thymus), recombinant human histone H1.0 (New England Biolabs, M2501), or recombinant human histone H1.4 (MyBioSource, MBS963223) and 1 μg of purified HeLa mononucleosomes (EpiCypher, 16-0002) at 4 °C with rotation for 12 h. Histone H1 was treated with λ-phosphatase for 20 min according to the manufacturer's protocol or phosphorylated for 30 min in kinase assay buffer as described below.

    Affinity Chromatography:

    Article Title: The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.
    Article Snippet: Cell pellets were resuspended in a binding buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-Mercaptoethanol] supplemented with protease inhibitors cocktail (Sigma, St. Louis, MO, USA). .. After being resuspended, cells were lysed via sonication and then debris was removed by centrifugation for 30 min at 11,000 g. The protein in the supernatant was purified using amylose affinity chromatography (New England BioLabs, Ipswich, MA), and then concentrated using Amicon Ultra-15 filters (Millipore, Schwalbach, Germany).

    Immunoprecipitation:

    Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans
    Article Snippet: At 48 h posttransfection (no stably integrated cell lines could be established for these two proteins), nuclear extracts were prepared, adjusted to 150 mM NaCl, and immunoprecipitated as follows. .. The beads were then washed three times with binding buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 0.1% NP-40, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors), and bound proteins were eluted in binding buffer supplemented with 400 μg of 3X FLAG peptide (Sigma)/ml for 4 h at 4°C in batch.

    Protease Inhibitor:

    Article Title: A Novel, Single, Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel Protein 1
    Article Snippet: .. Testis membrane protein (∼1 mg) was solubilized with 1% Triton X-100 in 250 μl of binding buffer (50 mM Na2 HPO4 , 300 mM NaCl, and 1× protease inhibitor cocktail [pH 8.0]) at 4°C for 1 h. Soluble protein was added to 100 μl of buffer-equilibrated cobalt resin (Clontech) supplemented with 20 mM imidazole (pH 7.5) in 250 μl of binding buffer and mixed at 4°C for 2 h. Following a quick spinning of the protein-resin mixture, the supernatant (flow through) was collected and concentrated with a Microcon-50 ultrafiltration column (Millipore, Bedford, MA), and the cobalt resin was washed twice with binding buffer. .. Bound protein was eluted with 300 mM imidazole in binding buffer.

    Positron Emission Tomography:

    Article Title: Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli
    Article Snippet: For the expression and purification of CEA fusion proteins, E. coli BL21 (DE3) strains (Novagen) were transformed with either pET-CEA or pET-Tat-CEA. .. Bacterial cells harvested by centrifugation at 1000 g for 10 min were resuspended in binding buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1·5% Triton X-100 (Sigma) and then disrupted by sonication on ice for 5 min. Sonicated lysates were centrifuged at 1600 g for 20 min at 4°C, and then the pellets containing CEA or Tat-CEA protein were resuspended in binding buffer containing 6 M urea (Sigma), sonicated and centrifuged at 1600 g for 30 min at 4°C.

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  • 99
    Millipore annexin v binding buffer
    HIF-1α knockdown by siRNA affects the cytotoxicity and autophagy of HEK-293 cells.  a  Transfection efficacy was verified by western blot analysis. HIF-1α protein expression in HEK-293 cells transfected with control or HIF-1α siRNA for 24 h.  b  Cell viability in the absence or presence of HIF-1α siRNA in HEK-293 cells.  c  Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry.  d  Quantification of AVOs with AO-stained cells transfected with control or HIF-1α siRNA using flow cytometry. The cells were transfected with control or HIF-1α siRNA for 24 h and treated with medium alone or 20 μg/ml ZnO NPs for 24 h. * p
    Annexin V Binding Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/annexin v binding buffer/product/Millipore
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    annexin v binding buffer - by Bioz Stars, 2020-04
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    87
    Millipore aptamer binding buffer
    Comparison between SRM assays of endogenous RelA in crude or <t>aptamer-enriched</t> fractions. RelA was measured in crude cellular fractions prepared from TNFα-stimulated A549 cells (A,B) or cellular fractions subjected to aptamer-enrichment (C,D).
    Aptamer Binding Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore 7 aad binding buffer
    Apoptotic response induced by 5-Fu and Gyp. (A) Cell apoptosis was detected by Annexin <t>V-PE/7-AAD</t> assay after 5-Fu and / or Gyp treatment for 24 h in SW-480 cells. (B) The expression level of caspase 3, caspase 9, Bcl-2 and cleaved-PARP in SW-480 cells was detected by western blotting. (C) Nuclear condensation and cell morphology change in SW-480 cells after 5-Fu co-treated with Gyp was observed using Hoechst 33342 staining. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p *
    7 Aad Binding Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HIF-1α knockdown by siRNA affects the cytotoxicity and autophagy of HEK-293 cells.  a  Transfection efficacy was verified by western blot analysis. HIF-1α protein expression in HEK-293 cells transfected with control or HIF-1α siRNA for 24 h.  b  Cell viability in the absence or presence of HIF-1α siRNA in HEK-293 cells.  c  Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry.  d  Quantification of AVOs with AO-stained cells transfected with control or HIF-1α siRNA using flow cytometry. The cells were transfected with control or HIF-1α siRNA for 24 h and treated with medium alone or 20 μg/ml ZnO NPs for 24 h. * p

    Journal: Particle and Fibre Toxicology

    Article Title: The role of hypoxia-inducible factor-1α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo

    doi: 10.1186/s12989-016-0163-3

    Figure Lengend Snippet: HIF-1α knockdown by siRNA affects the cytotoxicity and autophagy of HEK-293 cells. a Transfection efficacy was verified by western blot analysis. HIF-1α protein expression in HEK-293 cells transfected with control or HIF-1α siRNA for 24 h. b Cell viability in the absence or presence of HIF-1α siRNA in HEK-293 cells. c Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. d Quantification of AVOs with AO-stained cells transfected with control or HIF-1α siRNA using flow cytometry. The cells were transfected with control or HIF-1α siRNA for 24 h and treated with medium alone or 20 μg/ml ZnO NPs for 24 h. * p

    Article Snippet: Cells were resuspended in 100 μl of 1 × Annexin V binding buffer (10 mM HEPES (pH 7.4), 0.14 M NaCl and 2.5 mM CaCl2 ) that contained 2 μl of Annexin V-FITC (Calbiochem, CA, USA) alone or in combination with 10 μl of PI (50 μg/ml) and were incubated in the dark at room temperature for 15 min.

    Techniques: Transfection, Western Blot, Expressing, Staining, Flow Cytometry, Cytometry

    Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. * p

    Journal: Particle and Fibre Toxicology

    Article Title: The role of hypoxia-inducible factor-1α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo

    doi: 10.1186/s12989-016-0163-3

    Figure Lengend Snippet: Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. * p

    Article Snippet: Cells were resuspended in 100 μl of 1 × Annexin V binding buffer (10 mM HEPES (pH 7.4), 0.14 M NaCl and 2.5 mM CaCl2 ) that contained 2 μl of Annexin V-FITC (Calbiochem, CA, USA) alone or in combination with 10 μl of PI (50 μg/ml) and were incubated in the dark at room temperature for 15 min.

    Techniques: Staining, Flow Cytometry, Cytometry, Incubation

    Large scale purification of bacterial IgG. (A) Non-reducing coomassie gel of peak fractions from Protein A HPLC purification of cell lysate. Left panel: 4G2 fractions 5 to 11 (F5 to F11). Right panel ET149 fractions 10 to 16 (F10 to F16). 30 µl of each fraction was run on the gel. A sample of the wash (LW) was run and shows no contaminants present indicating the column was sufficiently washed to remove non-binding proteins. Fully assembled IgGs (2H2L) are indicated. (B). Reducing coomassie gels (4G2-F7, ET149-F12 13) and adjacent immunoblots (I) showing representative fractions from each Protein A elution (indicated with * on panel A). 30 µl of each fraction was loaded for coomassie and 3.75 µl for immunoblot. Blots were probed with both anti-IgG Fc and anti-Kappa chain polyclonals showing that majority of protein bands in the fraction are neither IgG heavy chain nor light chain. A separate reducing coomassie gel shows 2 µg of bacterial IgG pooled eluate (EL) after Protein L purification showing successful removal of the contaminating proteins and degraded heavy chain fragments. The equivalent amount of mammalian cell culture-derived IgG (IgG) was loaded for comparison. Individual heavy (HC) and light (LC) chains are indicated although the light chain for ET149 appears as two separate species.

    Journal: PLoS ONE

    Article Title: Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

    doi: 10.1371/journal.pone.0010261

    Figure Lengend Snippet: Large scale purification of bacterial IgG. (A) Non-reducing coomassie gel of peak fractions from Protein A HPLC purification of cell lysate. Left panel: 4G2 fractions 5 to 11 (F5 to F11). Right panel ET149 fractions 10 to 16 (F10 to F16). 30 µl of each fraction was run on the gel. A sample of the wash (LW) was run and shows no contaminants present indicating the column was sufficiently washed to remove non-binding proteins. Fully assembled IgGs (2H2L) are indicated. (B). Reducing coomassie gels (4G2-F7, ET149-F12 13) and adjacent immunoblots (I) showing representative fractions from each Protein A elution (indicated with * on panel A). 30 µl of each fraction was loaded for coomassie and 3.75 µl for immunoblot. Blots were probed with both anti-IgG Fc and anti-Kappa chain polyclonals showing that majority of protein bands in the fraction are neither IgG heavy chain nor light chain. A separate reducing coomassie gel shows 2 µg of bacterial IgG pooled eluate (EL) after Protein L purification showing successful removal of the contaminating proteins and degraded heavy chain fragments. The equivalent amount of mammalian cell culture-derived IgG (IgG) was loaded for comparison. Individual heavy (HC) and light (LC) chains are indicated although the light chain for ET149 appears as two separate species.

    Article Snippet: Peak fractions were pooled and buffer exchanged into 2 ml Protein L binding buffer (100 mM sodium phosphate buffer pH 7.2, 150 mM NaCl) with a Amicon 30000 MWCO concentrator (Millipore, United States) and then bound to 400 µl Protein L agarose bead 50% slurry (Pierce, United States) overnight at 4°C.

    Techniques: Purification, High Performance Liquid Chromatography, Binding Assay, Western Blot, Cell Culture, Derivative Assay

    Comparison between bacterial and mammalian IgG. (A). Non-reducing SDS-PAGE of 2 µg of pooled bacterial IgG eluate after protein L purification (EL) and mammalian cell culture-derived IgG (IgG) indicates that the majority of purified IgG was fully assembled IgG (2H2L) although partially assembled IgG (2H1L, 1H1L) are also present. (B). Direct binding ELISA of serially diluted bacterial (-▪-)- and mammalian cell culture (-•-)-derived IgG against Dengue serotype-2 virus and epsilon toxin showing similar binding curves. Binding of antibody was detected using anti-Human IgG Fc-HRPO as the secondary antibody.

    Journal: PLoS ONE

    Article Title: Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

    doi: 10.1371/journal.pone.0010261

    Figure Lengend Snippet: Comparison between bacterial and mammalian IgG. (A). Non-reducing SDS-PAGE of 2 µg of pooled bacterial IgG eluate after protein L purification (EL) and mammalian cell culture-derived IgG (IgG) indicates that the majority of purified IgG was fully assembled IgG (2H2L) although partially assembled IgG (2H1L, 1H1L) are also present. (B). Direct binding ELISA of serially diluted bacterial (-▪-)- and mammalian cell culture (-•-)-derived IgG against Dengue serotype-2 virus and epsilon toxin showing similar binding curves. Binding of antibody was detected using anti-Human IgG Fc-HRPO as the secondary antibody.

    Article Snippet: Peak fractions were pooled and buffer exchanged into 2 ml Protein L binding buffer (100 mM sodium phosphate buffer pH 7.2, 150 mM NaCl) with a Amicon 30000 MWCO concentrator (Millipore, United States) and then bound to 400 µl Protein L agarose bead 50% slurry (Pierce, United States) overnight at 4°C.

    Techniques: SDS Page, Purification, Cell Culture, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Comparison between SRM assays of endogenous RelA in crude or aptamer-enriched fractions. RelA was measured in crude cellular fractions prepared from TNFα-stimulated A549 cells (A,B) or cellular fractions subjected to aptamer-enrichment (C,D).

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantification of Activated NF-?B/RelA Complexes Using ssDNA Aptamer Affinity - Stable Isotope Dilution--Selected Reaction Monitoring--Mass Spectrometry *

    doi: 10.1074/mcp.M111.008771

    Figure Lengend Snippet: Comparison between SRM assays of endogenous RelA in crude or aptamer-enriched fractions. RelA was measured in crude cellular fractions prepared from TNFα-stimulated A549 cells (A,B) or cellular fractions subjected to aptamer-enrichment (C,D).

    Article Snippet: Screening was performed by incubating GST-RelA protein with the aptamer pool in aptamer binding buffer (20 m m Tris-HCl pH 7.4, 150 m m NaCl and 1 m m MgCl2 ) for 30–60 min at room temperature, followed by separation of the free DNA from the bound DNA by filtering through a 25 mm diameter nitrocellulose filter (Millipore, Billerica, MA).

    Techniques:

    Isolation of ssDNA aptamer binding to RelA (1–313). A , Sequences of P028F4 and nonspecific P028A1. Small case, flanking sequences. B , Sensorgram tracings for GST-RelA binding to P028F4. Increasing concentrations of GST-RelA (indicated in the inset)

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantification of Activated NF-?B/RelA Complexes Using ssDNA Aptamer Affinity - Stable Isotope Dilution--Selected Reaction Monitoring--Mass Spectrometry *

    doi: 10.1074/mcp.M111.008771

    Figure Lengend Snippet: Isolation of ssDNA aptamer binding to RelA (1–313). A , Sequences of P028F4 and nonspecific P028A1. Small case, flanking sequences. B , Sensorgram tracings for GST-RelA binding to P028F4. Increasing concentrations of GST-RelA (indicated in the inset)

    Article Snippet: Screening was performed by incubating GST-RelA protein with the aptamer pool in aptamer binding buffer (20 m m Tris-HCl pH 7.4, 150 m m NaCl and 1 m m MgCl2 ) for 30–60 min at room temperature, followed by separation of the free DNA from the bound DNA by filtering through a 25 mm diameter nitrocellulose filter (Millipore, Billerica, MA).

    Techniques: Isolation, Binding Assay

    Schematic diagram of the SRM assay of aptamer-enriched RelA and its associated proteins. First, biotin-conjugated RelA specific aptamer was incubated with crude cell extract. Second, the Bt-aptamer RelA was captured by SA-coated magnetic beads. The beads

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantification of Activated NF-?B/RelA Complexes Using ssDNA Aptamer Affinity - Stable Isotope Dilution--Selected Reaction Monitoring--Mass Spectrometry *

    doi: 10.1074/mcp.M111.008771

    Figure Lengend Snippet: Schematic diagram of the SRM assay of aptamer-enriched RelA and its associated proteins. First, biotin-conjugated RelA specific aptamer was incubated with crude cell extract. Second, the Bt-aptamer RelA was captured by SA-coated magnetic beads. The beads

    Article Snippet: Screening was performed by incubating GST-RelA protein with the aptamer pool in aptamer binding buffer (20 m m Tris-HCl pH 7.4, 150 m m NaCl and 1 m m MgCl2 ) for 30–60 min at room temperature, followed by separation of the free DNA from the bound DNA by filtering through a 25 mm diameter nitrocellulose filter (Millipore, Billerica, MA).

    Techniques: Incubation, Magnetic Beads

    Isolation of RelA(1–313) Binding Single-stranded Aptamer

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantification of Activated NF-?B/RelA Complexes Using ssDNA Aptamer Affinity - Stable Isotope Dilution--Selected Reaction Monitoring--Mass Spectrometry *

    doi: 10.1074/mcp.M111.008771

    Figure Lengend Snippet: Isolation of RelA(1–313) Binding Single-stranded Aptamer

    Article Snippet: Screening was performed by incubating GST-RelA protein with the aptamer pool in aptamer binding buffer (20 m m Tris-HCl pH 7.4, 150 m m NaCl and 1 m m MgCl2 ) for 30–60 min at room temperature, followed by separation of the free DNA from the bound DNA by filtering through a 25 mm diameter nitrocellulose filter (Millipore, Billerica, MA).

    Techniques: Isolation, Binding Assay

    Apoptotic response induced by 5-Fu and Gyp. (A) Cell apoptosis was detected by Annexin V-PE/7-AAD assay after 5-Fu and / or Gyp treatment for 24 h in SW-480 cells. (B) The expression level of caspase 3, caspase 9, Bcl-2 and cleaved-PARP in SW-480 cells was detected by western blotting. (C) Nuclear condensation and cell morphology change in SW-480 cells after 5-Fu co-treated with Gyp was observed using Hoechst 33342 staining. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p *

    Journal: PLoS ONE

    Article Title: Gypenosides Synergistically Enhances the Anti-Tumor Effect of 5-Fluorouracil on Colorectal Cancer In Vitro and In Vivo: A Role for Oxidative Stress-Mediated DNA Damage and p53 Activation

    doi: 10.1371/journal.pone.0137888

    Figure Lengend Snippet: Apoptotic response induced by 5-Fu and Gyp. (A) Cell apoptosis was detected by Annexin V-PE/7-AAD assay after 5-Fu and / or Gyp treatment for 24 h in SW-480 cells. (B) The expression level of caspase 3, caspase 9, Bcl-2 and cleaved-PARP in SW-480 cells was detected by western blotting. (C) Nuclear condensation and cell morphology change in SW-480 cells after 5-Fu co-treated with Gyp was observed using Hoechst 33342 staining. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p *

    Article Snippet: After that 100 μl cells of each sample was suspended in a mixture of 100 μl Annexin V-PE and 7-AAD binding buffer, incubated at 37°C for 20 min in the dark and subjected to flow cytometry analysis (Millipore, USA) immediately.

    Techniques: Expressing, Western Blot, Staining