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Nacalai ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic Acid, supplied by Nacalai, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ethylenediaminetetraacetic acid/product/Nacalai
Average 96 stars, based on 5 article reviews
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ethylenediaminetetraacetic acid - by Bioz Stars, 2020-04
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Centrifugation:

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Cytometry:

Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity
Article Snippet: .. Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). .. After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 (1 μg/ml) for 30 min at 4°C followed by incubation with Oregon green-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG (Thermo Fisher Scientific Inc.).

Blocking Assay:

Article Title: Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice
Article Snippet: Western blot analysis Cultured cells were lysed in RIPA buffer (1% Nonidet P-40, 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). .. The membrane was incubated in polyvinylidene blocking reagent (Toyobo, Tokyo, Japan) at room temperature for 20 min, and then incubated at 4 °C overnight with the appropriate primary antibody diluted in Can Get Signal Solution 1 (Toyobo).

SDS-Gel:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. The avidin-bound complexes were washed with the same buffer and subjected to SDS gel electrophoresis.

Incubation:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. Equal amounts of protein were subjected to SDS-PAGE or incubated with Streptavidin Sepharose 4B (GE Healthcare Biosciences, Piscataway, NJ) for 1 h to isolate biotinylated proteins.

Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity
Article Snippet: Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). .. After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 (1 μg/ml) for 30 min at 4°C followed by incubation with Oregon green-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG (Thermo Fisher Scientific Inc.).

Article Title: Phospholipid‐flipping activity of P4‐ ATPase drives membrane curvature
Article Snippet: Cells were lysed in lysis buffer (20 mM HEPES‐KOH [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% NP‐40) containing a protease inhibitor cocktail (Nacalai Tesque) for 20 min on ice. .. The membrane was blocked with 5% skimmed milk and sequentially incubated with the indicated primary and horseradish peroxidase‐conjugated secondary antibodies.

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. After incubation with the secondary antibodies, proteins were visualized using Chemi-Lumi One Super (Nacalai Tesque) and a LAS-1000 imaging system (Fujifilm, Tokyo, Japan).

Expressing:

Article Title: HIF-1-dependent lipin1 induction prevents excessive lipid accumulation in choline-deficient diet-induced fatty liver
Article Snippet: In some experiments, AML12 cells exposed to choline-deficient medium were lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% CHAPS, 5 mM EDTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and resultant cell lysates (15 μg) were used. .. The expression of PGC-1α, lipin1, HIF-1α, lamin, and tubulin was detected using specific antibodies against the respective antigens as follows: PGC-1α (2178 S, Novus Biologicals; Littleton, CO, USA), lipin1 (AF3885, R & D Systems; Minneapolis, MN, USA), HIF-1α (36169 and 14179, Cell Signaling Technology, Danvers, MA, USA), lamin A/C (sc-20681, Santa Cruz Biotechnology; Santa Cruz, CA, USA), and tubulin (T9026, Sigma-Aldrich, St. Louis, MO, USA).

Western Blot:

Article Title: HIF-1-dependent lipin1 induction prevents excessive lipid accumulation in choline-deficient diet-induced fatty liver
Article Snippet: Paragraph title: Western blotting ... In some experiments, AML12 cells exposed to choline-deficient medium were lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% CHAPS, 5 mM EDTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and resultant cell lysates (15 μg) were used.

Article Title: Selective inhibition of the kinase DYRK1A by targeting its folding process
Article Snippet: Immunoprecipitation of FLAG-DYRK1A HEK293 cells were cultured in the presence of 1 μg ml−1 of doxycycline, 1 μM of Shield-1, 1 μM of epoxomicin and a proteasome inhibitor (Peptide Institute), with or without 10 μM of small molecules for 5 h. The cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% Empigen BB and protease/phosphatase inhibitor cocktails (Nacalai Tesque). .. The clarified cell lysates were processed with anti-FLAG (clone M2) beads (F2426, Sigma-Aldrich) and the bound proteins were then eluted with SDS-urea buffer, then analysed with SDS–PAGE followed by western blot analysis ( ).

Article Title: Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice
Article Snippet: .. Western blot analysis Cultured cells were lysed in RIPA buffer (1% Nonidet P-40, 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). .. Equal amount of lysates were separated by 4–15% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA).

Article Title: Glypican-1 targeted antibody-based therapy induces preclinical antitumor activity against esophageal squamous cell carcinoma
Article Snippet: Digestion with heparinase III Proteins were extracted from fresh-frozen samples of ESCC with 50 mM Tris-HCl (pH 7.4),150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% protease inhibitor cocktail (Nacalai Tesque) and 1% phosphatase-inhibitor cocktail (Nacalai Tesque). .. Protein extracts from ESCC tissues or normal tissues were digested with Heparinase III, as described previously [ ], and used for western blotting.

Article Title: Type I interferon protects neurons from prions in in vivo models
Article Snippet: The culture cells and animal tissues treated with various experimental conditions were lysed in 1 × lysis buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% Triton™ X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and protease inhibitors (Nacalai Tesque)], for 30 min at 4°C. .. Bands were detected by appropriate primary antibodies and horseradish peroxidase-labelled secondary antibodies, and visualized using the Chemi-Lumi One L (Nacalai Tesque), ECL prime Western Blotting Detection Kit (GE Healthcare Life Sciences), or Clarity™ Western ECL Substrate (Bio-Rad) to get appropriate results by sufficient enzymatic reaction.

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: .. Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. Nuclear fractions of cells and kidney tissues were prepared using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit (Wako) according to the manufacturer’s protocol.

Flow Cytometry:

Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity
Article Snippet: .. Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). .. After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 (1 μg/ml) for 30 min at 4°C followed by incubation with Oregon green-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG (Thermo Fisher Scientific Inc.).

Transfection:

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. HEK293T cells or NIH3T3 cells were transfected with plasmids by using the FuGene HD transfection reagent (Roche).

Protease Inhibitor:

Article Title: Phospholipid‐flipping activity of P4‐ ATPase drives membrane curvature
Article Snippet: .. Cells were lysed in lysis buffer (20 mM HEPES‐KOH [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% NP‐40) containing a protease inhibitor cocktail (Nacalai Tesque) for 20 min on ice. ..

Article Title: Glypican-1 targeted antibody-based therapy induces preclinical antitumor activity against esophageal squamous cell carcinoma
Article Snippet: .. Digestion with heparinase III Proteins were extracted from fresh-frozen samples of ESCC with 50 mM Tris-HCl (pH 7.4),150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% protease inhibitor cocktail (Nacalai Tesque) and 1% phosphatase-inhibitor cocktail (Nacalai Tesque). .. The total proteins from adult human normal heart, kidney, small intestine, colon tissues were purchased from BioChain Institute, Inc (Hayward,CA, USA).

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: .. Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. Nuclear fractions of cells and kidney tissues were prepared using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit (Wako) according to the manufacturer’s protocol.

Cell Culture:

Article Title: Selective inhibition of the kinase DYRK1A by targeting its folding process
Article Snippet: .. Immunoprecipitation of FLAG-DYRK1A HEK293 cells were cultured in the presence of 1 μg ml−1 of doxycycline, 1 μM of Shield-1, 1 μM of epoxomicin and a proteasome inhibitor (Peptide Institute), with or without 10 μM of small molecules for 5 h. The cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% Empigen BB and protease/phosphatase inhibitor cocktails (Nacalai Tesque). .. The clarified cell lysates were processed with anti-FLAG (clone M2) beads (F2426, Sigma-Aldrich) and the bound proteins were then eluted with SDS-urea buffer, then analysed with SDS–PAGE followed by western blot analysis ( ).

Article Title: Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice
Article Snippet: .. Western blot analysis Cultured cells were lysed in RIPA buffer (1% Nonidet P-40, 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). .. Equal amount of lysates were separated by 4–15% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA).

Imaging:

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. After incubation with the secondary antibodies, proteins were visualized using Chemi-Lumi One Super (Nacalai Tesque) and a LAS-1000 imaging system (Fujifilm, Tokyo, Japan).

Recombinant:

Article Title: Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿
Article Snippet: EDTA and EGTA were purchased from Nacalai Tesque Inc. (Kyoto, Japan). .. Recombinant LLO ( > 95% purity) was purchased from Abcam (Cambridge, United Kingdom).

Nucleic Acid Electrophoresis:

Article Title: Type I interferon protects neurons from prions in in vivo models
Article Snippet: The culture cells and animal tissues treated with various experimental conditions were lysed in 1 × lysis buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% Triton™ X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and protease inhibitors (Nacalai Tesque)], for 30 min at 4°C. .. The lysates were then treated by sodium dodecyl sulphate (SDS) sample buffer and were separated with 15% SDS-polyacrylamide gel electrophoresis, and blotted to a polyvinylidene difluoride membrane.

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. Aliquots of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a reduced condition and transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA), which was probed with the primary antibodies.

In Vivo:

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Fluorescence:

Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity
Article Snippet: Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). .. Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Isolation:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: .. After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. Equal amounts of protein were subjected to SDS-PAGE or incubated with Streptavidin Sepharose 4B (GE Healthcare Biosciences, Piscataway, NJ) for 1 h to isolate biotinylated proteins.

Avidin-Biotin Assay:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. The avidin-bound complexes were washed with the same buffer and subjected to SDS gel electrophoresis.

Pyrolysis Gas Chromatography:

Article Title: HIF-1-dependent lipin1 induction prevents excessive lipid accumulation in choline-deficient diet-induced fatty liver
Article Snippet: In some experiments, AML12 cells exposed to choline-deficient medium were lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% CHAPS, 5 mM EDTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and resultant cell lysates (15 μg) were used. .. The expression of PGC-1α, lipin1, HIF-1α, lamin, and tubulin was detected using specific antibodies against the respective antigens as follows: PGC-1α (2178 S, Novus Biologicals; Littleton, CO, USA), lipin1 (AF3885, R & D Systems; Minneapolis, MN, USA), HIF-1α (36169 and 14179, Cell Signaling Technology, Danvers, MA, USA), lamin A/C (sc-20681, Santa Cruz Biotechnology; Santa Cruz, CA, USA), and tubulin (T9026, Sigma-Aldrich, St. Louis, MO, USA).

SDS Page:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. Equal amounts of protein were subjected to SDS-PAGE or incubated with Streptavidin Sepharose 4B (GE Healthcare Biosciences, Piscataway, NJ) for 1 h to isolate biotinylated proteins.

Article Title: Selective inhibition of the kinase DYRK1A by targeting its folding process
Article Snippet: Immunoprecipitation of FLAG-DYRK1A HEK293 cells were cultured in the presence of 1 μg ml−1 of doxycycline, 1 μM of Shield-1, 1 μM of epoxomicin and a proteasome inhibitor (Peptide Institute), with or without 10 μM of small molecules for 5 h. The cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% Empigen BB and protease/phosphatase inhibitor cocktails (Nacalai Tesque). .. The clarified cell lysates were processed with anti-FLAG (clone M2) beads (F2426, Sigma-Aldrich) and the bound proteins were then eluted with SDS-urea buffer, then analysed with SDS–PAGE followed by western blot analysis ( ).

Article Title: Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice
Article Snippet: Western blot analysis Cultured cells were lysed in RIPA buffer (1% Nonidet P-40, 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). .. Equal amount of lysates were separated by 4–15% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA).

Article Title: Phospholipid‐flipping activity of P4‐ ATPase drives membrane curvature
Article Snippet: Cells were lysed in lysis buffer (20 mM HEPES‐KOH [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% NP‐40) containing a protease inhibitor cocktail (Nacalai Tesque) for 20 min on ice. .. Proteins (30 μg) were separated by SDS–PAGE and electroblotted onto an Immobilon‐P transfer membrane (Millipore EMD).

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Software:

Article Title: Type I interferon protects neurons from prions in in vivo models
Article Snippet: The culture cells and animal tissues treated with various experimental conditions were lysed in 1 × lysis buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% Triton™ X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and protease inhibitors (Nacalai Tesque)], for 30 min at 4°C. .. Band intensities were quantified using ImageJ software (NIH).

Electrophoresis:

Article Title: Basolateral BMP Signaling in Polarized Epithelial Cells
Article Snippet: After cell-surface biotinylation was performed from either apical or basolateral sides, using the Pierce Cell Surface Protein Isolation Kit (Thermo Scientific, Rockford, IL), the cells were scraped with a rubber policeman in buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and centrifuged at 15000 rpm for 20 min at 4°C. .. The avidin-bound complexes were washed with the same buffer and subjected to SDS gel electrophoresis.

Article Title: Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures
Article Snippet: .. Tris-HCl, EDTA and MgCl2 for electrophoresis analysis were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). .. Water was deionized (18.0 MΩ cm specific resistance) by a Milli-Q system (Millipore Corp., Bedford, MA).

Co-Immunoprecipitation Assay:

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

In Vitro:

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Immunoprecipitation:

Article Title: Selective inhibition of the kinase DYRK1A by targeting its folding process
Article Snippet: .. Immunoprecipitation of FLAG-DYRK1A HEK293 cells were cultured in the presence of 1 μg ml−1 of doxycycline, 1 μM of Shield-1, 1 μM of epoxomicin and a proteasome inhibitor (Peptide Institute), with or without 10 μM of small molecules for 5 h. The cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% Empigen BB and protease/phosphatase inhibitor cocktails (Nacalai Tesque). .. The clarified cell lysates were processed with anti-FLAG (clone M2) beads (F2426, Sigma-Aldrich) and the bound proteins were then eluted with SDS-urea buffer, then analysed with SDS–PAGE followed by western blot analysis ( ).

Lysis:

Article Title: Phospholipid‐flipping activity of P4‐ ATPase drives membrane curvature
Article Snippet: .. Cells were lysed in lysis buffer (20 mM HEPES‐KOH [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% NP‐40) containing a protease inhibitor cocktail (Nacalai Tesque) for 20 min on ice. ..

Article Title: Type I interferon protects neurons from prions in in vivo models
Article Snippet: .. The culture cells and animal tissues treated with various experimental conditions were lysed in 1 × lysis buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% Triton™ X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and protease inhibitors (Nacalai Tesque)], for 30 min at 4°C. .. The lysates were then treated by sodium dodecyl sulphate (SDS) sample buffer and were separated with 15% SDS-polyacrylamide gel electrophoresis, and blotted to a polyvinylidene difluoride membrane.

Article Title: Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction
Article Snippet: .. Coimmunoprecipitation assay in vitro and in vivo E13.5 spinal cords and DRG dissected and lysed with Triton-X100 lysis buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM EDTA, and 1% Triton-X100) containing protease inhibitor cocktail (Nacalai, Japan) and incubated on ice for 1 h. The lysate was centrifuged at 14,000 rpm for 10 minutes at 4C, and the supernatant was precleared with protein G–conjugated Sepharose beads (GE Healthcare) for 1 h at 4C, followed by incubation with the anti-EphA4 antibody (Zymed) or control mouse IgG overnight at 4C. .. Protein G-conjugated Sepharose beads were added and washed several times after centrifugation, and then SDS-PAGE was performed.

Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response
Article Snippet: .. Western Blotting Total lysates of cells and kidney tissues were obtained by lysis in RIPA Buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 20 mg/mL Na3VO4, 10 mM NaF, and 1 mM PMSF) with a protease inhibitor cocktail (Nacalai Tesque). .. Nuclear fractions of cells and kidney tissues were prepared using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit (Wako) according to the manufacturer’s protocol.

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    Nacalai edta
    Surface modification of the lattice. ( a ) Design of a cross-shaped DNA origami structure carrying four biotinylated staple strands (yellow dots). The top face of the origami has four biotin moieties. ( b ) Time-lapse AFM images of the modification of the lattice with streptavidin molecules. While scanning of the same area was ongoing, 15 μl of the folding buffer containing 20 μM streptavidin was injected into 135 μl of the standard buffer (20 mM <t>Tris</t> buffer (pH 7.6), 1 mM <t>EDTA</t> and 10 mM MgCl 2 ), so that the final concentration of streptavidin was 2 μM. Images were obtained at a scan rate of 0.2 frames per s. The elapsed time is shown in each image. The solution of streptavidin was added at 35 s. Details are seen in Supplementary Movie 3 . ( c ) Design of a cross-shaped DNA origami structure carrying eight biotinylated staple strands (yellow dots). ( d , e ) AFM images of the lattice after modification with streptavidin (2 μM). ( f ) A topographic AFM image of the streptavidin-modified DNA origami lattice. Scale bars, 100 nm.
    Edta, supplied by Nacalai, used in various techniques. Bioz Stars score: 94/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Surface modification of the lattice. ( a ) Design of a cross-shaped DNA origami structure carrying four biotinylated staple strands (yellow dots). The top face of the origami has four biotin moieties. ( b ) Time-lapse AFM images of the modification of the lattice with streptavidin molecules. While scanning of the same area was ongoing, 15 μl of the folding buffer containing 20 μM streptavidin was injected into 135 μl of the standard buffer (20 mM Tris buffer (pH 7.6), 1 mM EDTA and 10 mM MgCl 2 ), so that the final concentration of streptavidin was 2 μM. Images were obtained at a scan rate of 0.2 frames per s. The elapsed time is shown in each image. The solution of streptavidin was added at 35 s. Details are seen in Supplementary Movie 3 . ( c ) Design of a cross-shaped DNA origami structure carrying eight biotinylated staple strands (yellow dots). ( d , e ) AFM images of the lattice after modification with streptavidin (2 μM). ( f ) A topographic AFM image of the streptavidin-modified DNA origami lattice. Scale bars, 100 nm.

    Journal: Nature Communications

    Article Title: Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

    doi: 10.1038/ncomms9052

    Figure Lengend Snippet: Surface modification of the lattice. ( a ) Design of a cross-shaped DNA origami structure carrying four biotinylated staple strands (yellow dots). The top face of the origami has four biotin moieties. ( b ) Time-lapse AFM images of the modification of the lattice with streptavidin molecules. While scanning of the same area was ongoing, 15 μl of the folding buffer containing 20 μM streptavidin was injected into 135 μl of the standard buffer (20 mM Tris buffer (pH 7.6), 1 mM EDTA and 10 mM MgCl 2 ), so that the final concentration of streptavidin was 2 μM. Images were obtained at a scan rate of 0.2 frames per s. The elapsed time is shown in each image. The solution of streptavidin was added at 35 s. Details are seen in Supplementary Movie 3 . ( c ) Design of a cross-shaped DNA origami structure carrying eight biotinylated staple strands (yellow dots). ( d , e ) AFM images of the lattice after modification with streptavidin (2 μM). ( f ) A topographic AFM image of the streptavidin-modified DNA origami lattice. Scale bars, 100 nm.

    Article Snippet: Tris-HCl, EDTA and MgCl2 for electrophoresis analysis were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

    Techniques: Modification, Injection, Concentration Assay

    Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Journal: Infection and Immunity

    Article Title: Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿

    doi: 10.1128/IAI.01143-09

    Figure Lengend Snippet: Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Article Snippet: EDTA and EGTA were purchased from Nacalai Tesque Inc. (Kyoto, Japan).

    Techniques: Infection

    Flow cytometry of hPDPN-transfected cells using anti-hPDPN antibodies LpMab-7 and chLpMab-7 Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with LpMab-7 A. and chLpMab-7 B. followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800. C. The epitope mapping of chLpMab-7 by Western blot analysis. Total cell lysates of point mutants of hPDPN were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/ml mAbs (chLpMab-7 or NZ-8 against hPDPN, 1E5 against FLAG tag, AC-15 against β-actin) and then with peroxidase-conjugated secondary antibodies; the membrane was analyzed using a Sayaca-Imager.

    Journal: Oncotarget

    Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

    doi:

    Figure Lengend Snippet: Flow cytometry of hPDPN-transfected cells using anti-hPDPN antibodies LpMab-7 and chLpMab-7 Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with LpMab-7 A. and chLpMab-7 B. followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800. C. The epitope mapping of chLpMab-7 by Western blot analysis. Total cell lysates of point mutants of hPDPN were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/ml mAbs (chLpMab-7 or NZ-8 against hPDPN, 1E5 against FLAG tag, AC-15 against β-actin) and then with peroxidase-conjugated secondary antibodies; the membrane was analyzed using a Sayaca-Imager.

    Article Snippet: Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.).

    Techniques: Flow Cytometry, Cytometry, Transfection, Fluorescence, Western Blot, Blocking Assay, Incubation, FLAG-tag

    Flow cytometry of endogenous hPDPN-expressing cell lines using anti-hPDPN antibodies LpMab-7 and chLpMab-7 Cancer cells A. or normal cells B. were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800.

    Journal: Oncotarget

    Article Title: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

    doi:

    Figure Lengend Snippet: Flow cytometry of endogenous hPDPN-expressing cell lines using anti-hPDPN antibodies LpMab-7 and chLpMab-7 Cancer cells A. or normal cells B. were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800.

    Article Snippet: Flow cytometry Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.).

    Techniques: Flow Cytometry, Cytometry, Expressing, Fluorescence