ethylenediaminetetraacetic acid edta disodium salt  (Millipore)


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    Name:
    Ethylenediaminetetraacetic acid calcium disodium salt
    Description:

    Catalog Number:
    ed2sc
    Price:
    None
    Applications:
    Used to eliminate enzyme inhibition by traces of heavy metals, and to inhibit enzymes that require divalent cations as cofactors. As this form is already chelated with Ca2+, it is selective for divalent cations with greater affinity for EDTA, which it will exchange for Ca2+.
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    Structured Review

    Millipore ethylenediaminetetraacetic acid edta disodium salt
    Ethylenediaminetetraacetic acid calcium disodium salt

    https://www.bioz.com/result/ethylenediaminetetraacetic acid edta disodium salt/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ethylenediaminetetraacetic acid edta disodium salt - by Bioz Stars, 2020-03
    99/100 stars

    Images

    1) Product Images from "Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound"

    Article Title: Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30327-7

    Experimental diagram. Progressive demineralization each day. EDTA - Ethylenediaminetetraacetic Acid solutions; QUS - Quantitative Ultrasound; QCT - Quantitative Computed Tomography; ICP OES - coupled plasma optical emission spectrometry. Day 0 - integer bone. Demineralization occurs from Day 1 to Day 5.
    Figure Legend Snippet: Experimental diagram. Progressive demineralization each day. EDTA - Ethylenediaminetetraacetic Acid solutions; QUS - Quantitative Ultrasound; QCT - Quantitative Computed Tomography; ICP OES - coupled plasma optical emission spectrometry. Day 0 - integer bone. Demineralization occurs from Day 1 to Day 5.

    Techniques Used: Computed Tomography

    2) Product Images from "Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound"

    Article Title: Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30327-7

    Experimental diagram. Progressive demineralization each day. EDTA - Ethylenediaminetetraacetic Acid solutions; QUS - Quantitative Ultrasound; QCT - Quantitative Computed Tomography; ICP OES - coupled plasma optical emission spectrometry. Day 0 - integer bone. Demineralization occurs from Day 1 to Day 5.
    Figure Legend Snippet: Experimental diagram. Progressive demineralization each day. EDTA - Ethylenediaminetetraacetic Acid solutions; QUS - Quantitative Ultrasound; QCT - Quantitative Computed Tomography; ICP OES - coupled plasma optical emission spectrometry. Day 0 - integer bone. Demineralization occurs from Day 1 to Day 5.

    Techniques Used: Computed Tomography

    Related Articles

    Clone Assay:

    Article Title: Identification of Extended-Spectrum, AmpC, and Carbapenem- Hydrolyzing ?-Lactamases in Escherichia coli and Klebsiella pneumoniae by Disk Tests
    Article Snippet: The enzymes produced by previously unpublished strains were identified by isoelectric focusing, PCR amplification, cloning, and sequencing, as described previously ( , , , ). .. Plasmids were transferred to porin-deficient K. pneumoniae ), while 3-aminophenylboronic acid (APB) and EDTA disodium salt (EDTA) were obtained from Sigma (St. Louis, MO).

    Centrifugation:

    Article Title: Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study
    Article Snippet: The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad). .. The protein pellet was obtained by centrifugation at 15,800 × g for 30 min at 4°C.

    Amplification:

    Article Title: Identification of Extended-Spectrum, AmpC, and Carbapenem- Hydrolyzing ?-Lactamases in Escherichia coli and Klebsiella pneumoniae by Disk Tests
    Article Snippet: The enzymes produced by previously unpublished strains were identified by isoelectric focusing, PCR amplification, cloning, and sequencing, as described previously ( , , , ). .. Plasmids were transferred to porin-deficient K. pneumoniae ), while 3-aminophenylboronic acid (APB) and EDTA disodium salt (EDTA) were obtained from Sigma (St. Louis, MO).

    Filtration:

    Article Title: Remediation of heavy metal contaminated soil by asymmetrical alternating current electrochemistry
    Article Snippet: An electrochemical filtration device composed of two Ami-PC electrodes with a tissue paper between as a separator was put at the end of the plastic tube and connected to a power supply. .. In the first treatment cycle, 8 ml of 30 mM EDTA disodium salt (Sigma-Aldrich, 98.5–101.5%) solution was infused by a syringe pump to wash through the soil column and then filtrated by the AACE filter (Supplementary Fig. ).

    Synthesized:

    Article Title: Synthesis and evaluation of nanoglobule-cystamine-(Gd-DO3A), a biodegradable nanosized magnetic resonance contrast agent for dynamic contrast-enhanced magnetic resonance urography
    Article Snippet: Materials Gadolinium acetate (Alfa Aesar, Ward Hill, MA), ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma-Aldrich, St. Louis, MO), Gd(DTPA-BMA; GE Healthcare, Amersham, UK), Spectra/Por 6 dialysis membrane (molecular weight cutoff 2 kDa; Spectrum Laboratories, Rancho Dominguez, CA, USA) were used as purchased. .. 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-(acetic acid)-10-(acetic acid isothiocyanatocystamine monoamide) (DO3A-SS-NCS) was synthesized according to a reported method., The nu/nu mice were purchased from the NCI (Frederick, MD) and were cared under an animal protocol approved by the University of Utah Institutional Animal Care and Use Committee.

    Construct:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT. .. In this system, free 57/55 Co or radiocobalt chelated by EDTA migrate with the eluent front (Rf = 1.0) while the radiolabeled constructs remain on the application point (Rf = 0.0).

    Incubation:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: .. In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT. .. The ITLC strips (150–771 Dark Green, Tec-Control Chromatography strips from Biodex Medical Systems) were eluted with citric acid (0.2 M, pH 2.0).

    Article Title: Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study
    Article Snippet: Sample preparation for proteomic analysis MHCC97H cells were seeded in 100-mm culture dishes at 1 × 106 cells/dish (1 × 105 cells/mL), incubated overnight, and then treated with or without 500 μg/mL C. cicadae for 48 h. The cells were harvested by trypsinization, washed three times with PBS, and centrifuged in a Beckman Spinchron DLX (Beckman and Coulter) at 400 × g for 5 min. .. The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad).

    Mass Spectrometry:

    Article Title: Lack of nitric oxide- and guanosine 3?:5?-cyclic monophosphate-dependent regulation of ?-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts
    Article Snippet: For quantification of intracellular calcium, following-in-time images obtained at 340 and 380 nm excitation, emission 510 nm (time interval between two following-in-time images: 800 ms) were digitalized by an analogical/digital converter (256×256 pixels) and rationed on a pixel-by-pixel basis. .. L -NMMA, L -NOARG, hirudin, d-Phe-Pro-Arg chloromethylketone (D-PPAK), atrial natriuretic factor (ANF), bradykinin (Bk) and ethylenediaminetetraacetic acid (EDTA) disodium salt, were purchased from Sigma Chemical Co.; α-thrombin was obtained from Boehringer Mannheim; SNAP was purchased from Tocris Cookson Ltd. (Bristol, U.K.).

    Crystallization Assay:

    Article Title: Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo
    Article Snippet: To minimize crystallization of the docetaxel, the stock solution was further diluted to 2.4 mg/ml with injectable normal saline (Samyang Anipham, Seoul, Republic of Korea) right before injection. .. Whole blood was collected from the heart using a 1 ml syringe with 25 gauge pre-coated with 10% (w /v ) ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma) for the blood cell counts.

    High Performance Liquid Chromatography:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: After labeling with 55 Co, radiochemical yield was evaluated by radio HPLC as previously described . .. In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT.

    Chromatography:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT. .. The ITLC strips (150–771 Dark Green, Tec-Control Chromatography strips from Biodex Medical Systems) were eluted with citric acid (0.2 M, pH 2.0).

    Protease Inhibitor:

    Article Title: Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study
    Article Snippet: .. The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad). .. The superfluous salt in the extract was removed by incubation with trichloroacetic acid (TCA)-acetone solution [20% TCA (MERCK, Germany), 20 mmol/L DTT in acetone (MERCK, Germany)] for 4 h at −40°C.

    Cell Culture:

    Article Title: The ACh-induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells
    Article Snippet: Acetylcholine (ACh), phenylephrine, indomethacin, ATII, collagenase type I, elastase, antibiotic solutions and ethylene diaminetetraacetic acid (EDTA) disodium salt were obtained from Sigma. .. Cell culture plastic supports were purchased from Costar (Corning Costar Co., Costar Italia, Milan, Italy).

    Article Title: Lack of nitric oxide- and guanosine 3?:5?-cyclic monophosphate-dependent regulation of ?-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts
    Article Snippet: L -NMMA, L -NOARG, hirudin, d-Phe-Pro-Arg chloromethylketone (D-PPAK), atrial natriuretic factor (ANF), bradykinin (Bk) and ethylenediaminetetraacetic acid (EDTA) disodium salt, were purchased from Sigma Chemical Co.; α-thrombin was obtained from Boehringer Mannheim; SNAP was purchased from Tocris Cookson Ltd. (Bristol, U.K.). .. Cell culture plastic supports were purchased from Costar (Corning Costar Co., Costar Italia, Milan, Italy).

    Generated:

    Article Title: Remediation of heavy metal contaminated soil by asymmetrical alternating current electrochemistry
    Article Snippet: The asymmetrical alternating current was generated by GIGOL DG1022A. .. In the first treatment cycle, 8 ml of 30 mM EDTA disodium salt (Sigma-Aldrich, 98.5–101.5%) solution was infused by a syringe pump to wash through the soil column and then filtrated by the AACE filter (Supplementary Fig. ).

    Imaging:

    Article Title: Lack of nitric oxide- and guanosine 3?:5?-cyclic monophosphate-dependent regulation of ?-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts
    Article Snippet: Paragraph title: Imaging analysis of cytosolic intracellular calcium ... L -NMMA, L -NOARG, hirudin, d-Phe-Pro-Arg chloromethylketone (D-PPAK), atrial natriuretic factor (ANF), bradykinin (Bk) and ethylenediaminetetraacetic acid (EDTA) disodium salt, were purchased from Sigma Chemical Co.; α-thrombin was obtained from Boehringer Mannheim; SNAP was purchased from Tocris Cookson Ltd. (Bristol, U.K.).

    Polymerase Chain Reaction:

    Article Title: Identification of Extended-Spectrum, AmpC, and Carbapenem- Hydrolyzing ?-Lactamases in Escherichia coli and Klebsiella pneumoniae by Disk Tests
    Article Snippet: The enzymes produced by previously unpublished strains were identified by isoelectric focusing, PCR amplification, cloning, and sequencing, as described previously ( , , , ). .. Plasmids were transferred to porin-deficient K. pneumoniae ), while 3-aminophenylboronic acid (APB) and EDTA disodium salt (EDTA) were obtained from Sigma (St. Louis, MO).

    Injection:

    Article Title: Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo
    Article Snippet: At day 5, the animals were anaesthetized by intraperitoneal injection of 50 mg/kg pentobarbital solution (Entobar, Hanlim Pharmaceuticals Inc., Seoul, Republic of Korea). .. Whole blood was collected from the heart using a 1 ml syringe with 25 gauge pre-coated with 10% (w /v ) ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma) for the blood cell counts.

    Recombinant:

    Article Title: Disulfide structure of alfimeprase: A recombinant analog of fibrolase
    Article Snippet: Purified, recombinant alfimeprase (CAS registry number 259074-76-5) is from Amgen. .. Ethylenediaminetetraacetic acid (EDTA) disodium salt was purchased from Sigma Chemical Co.

    Molecular Weight:

    Article Title: Synthesis and evaluation of nanoglobule-cystamine-(Gd-DO3A), a biodegradable nanosized magnetic resonance contrast agent for dynamic contrast-enhanced magnetic resonance urography
    Article Snippet: .. Materials Gadolinium acetate (Alfa Aesar, Ward Hill, MA), ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma-Aldrich, St. Louis, MO), Gd(DTPA-BMA; GE Healthcare, Amersham, UK), Spectra/Por 6 dialysis membrane (molecular weight cutoff 2 kDa; Spectrum Laboratories, Rancho Dominguez, CA, USA) were used as purchased. ..

    Article Title: Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix
    Article Snippet: Linear PEG with nominal molecular weight of 3.4 kDa was purchased from Acros (Pittsburg, PA). .. Piperidine, calcium hydride, tetrahydrofuran (THF), trimethylsilane (TMS), triethylamine (TEA), tin (II) 2-ethylhexanoate (TOC), acryloyl chloride (AC), acrylic acid, dimethylsulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) disodium salt were purchased from Sigma-Aldrich (St. Louis, MO).

    Fluorescence:

    Article Title: Lack of nitric oxide- and guanosine 3?:5?-cyclic monophosphate-dependent regulation of ?-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts
    Article Snippet: In order to decrease cell light exposure which can decrease fura-2 fluorescence, the filter wheeler was stopped in a dark position and a ratio image was obtained every 3 s Calibration curves were performed using ionomycin (Calbiochem) and ethylenebis (oxyethylenenitrilo)-tetraacetic acid (EGTA, Aldrich-Chemi, Steinheim, Germany) as described ( ) and a dissociation constant for fura-2 of 224 n M . .. L -NMMA, L -NOARG, hirudin, d-Phe-Pro-Arg chloromethylketone (D-PPAK), atrial natriuretic factor (ANF), bradykinin (Bk) and ethylenediaminetetraacetic acid (EDTA) disodium salt, were purchased from Sigma Chemical Co.; α-thrombin was obtained from Boehringer Mannheim; SNAP was purchased from Tocris Cookson Ltd. (Bristol, U.K.).

    Isolation:

    Article Title: Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo
    Article Snippet: Whole blood was collected from the heart using a 1 ml syringe with 25 gauge pre-coated with 10% (w /v ) ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma) for the blood cell counts. .. The other half of the spleen and the whole thymus and femurs isolated from the mice were soaked and fixed in a neutral buffered formalin (pH 6.8–7.2, BBC Biochemical, Mount Vernon, WA, USA) for 72 h at 4 °C with gentle shaking.

    Article Title: A Possible Mechanism of Action of the Chemopreventive Effects of Sarcotriol on Skin Tumor Development in CD-1 Mice
    Article Snippet: General Sarcophine was isolated from the soft coral Sarcophyton glaucum collected from several locations of the Red Sea in Egypt. .. DMBA, TPA, calf thymus DNA, EDTA disodium salt were purchased from Sigma Chemical Co. (St. Louis, MO).

    Labeling:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: Paragraph title: Labeling and stability ... In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT.

    Mouse Assay:

    Article Title: Synthesis and evaluation of nanoglobule-cystamine-(Gd-DO3A), a biodegradable nanosized magnetic resonance contrast agent for dynamic contrast-enhanced magnetic resonance urography
    Article Snippet: Materials Gadolinium acetate (Alfa Aesar, Ward Hill, MA), ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma-Aldrich, St. Louis, MO), Gd(DTPA-BMA; GE Healthcare, Amersham, UK), Spectra/Por 6 dialysis membrane (molecular weight cutoff 2 kDa; Spectrum Laboratories, Rancho Dominguez, CA, USA) were used as purchased. .. 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-(acetic acid)-10-(acetic acid isothiocyanatocystamine monoamide) (DO3A-SS-NCS) was synthesized according to a reported method., The nu/nu mice were purchased from the NCI (Frederick, MD) and were cared under an animal protocol approved by the University of Utah Institutional Animal Care and Use Committee.

    Article Title: Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo
    Article Snippet: Then, mice orally received EPR (Group 3, 30 mg/kg; Group 4, 100 mg/kg; Group 5, 300 mg/kg; n = 10 mice/group) or 0.5% (w /v ) carboxymethylcellulose (CMC, Group 2, n = 10 mice, Sigma, St. Louis, MO, USA) as a vehicle for 4 days. .. Whole blood was collected from the heart using a 1 ml syringe with 25 gauge pre-coated with 10% (w /v ) ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma) for the blood cell counts.

    Sequencing:

    Article Title: Identification of Extended-Spectrum, AmpC, and Carbapenem- Hydrolyzing ?-Lactamases in Escherichia coli and Klebsiella pneumoniae by Disk Tests
    Article Snippet: The enzymes produced by previously unpublished strains were identified by isoelectric focusing, PCR amplification, cloning, and sequencing, as described previously ( , , , ). .. Plasmids were transferred to porin-deficient K. pneumoniae ), while 3-aminophenylboronic acid (APB) and EDTA disodium salt (EDTA) were obtained from Sigma (St. Louis, MO).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT. .. The system was previously validated by radio-HPLC and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Protective Effects of Simvastatin, a Lipid Lowering Agent, against Oxidative Damage in Experimental Diabetic Rats
    Article Snippet: Chemicals Simvastatin (SMV), Streptozotocin (STZ), hydrogen peroxide, glutathione, 5,5′-dithio-bis -(2-nitrobenzoic acid), thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane, glutathione-reduced form, glutathione reductase, oxidized glutathione (GSSG), NADPH-tetra salt, and ethylenediamine tetra acetic acid (EDTA) disodium salt were purchased from Sigma-Aldrich Chemical (St Louis, MO, USA). .. Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) reagent assay kits were purchased from Cayman chemical (MI, USA).

    Purification:

    Article Title: Disulfide structure of alfimeprase: A recombinant analog of fibrolase
    Article Snippet: Purified, recombinant alfimeprase (CAS registry number 259074-76-5) is from Amgen. .. Ethylenediaminetetraacetic acid (EDTA) disodium salt was purchased from Sigma Chemical Co.

    SDS Page:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT. .. The system was previously validated by radio-HPLC and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Electron Paramagnetic Resonance:

    Article Title: Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo
    Article Snippet: Then, mice orally received EPR (Group 3, 30 mg/kg; Group 4, 100 mg/kg; Group 5, 300 mg/kg; n = 10 mice/group) or 0.5% (w /v ) carboxymethylcellulose (CMC, Group 2, n = 10 mice, Sigma, St. Louis, MO, USA) as a vehicle for 4 days. .. Whole blood was collected from the heart using a 1 ml syringe with 25 gauge pre-coated with 10% (w /v ) ethylenediaminetetraacetic acid (EDTA) dipotassium salt (Sigma) for the blood cell counts.

    Sample Prep:

    Article Title: Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study
    Article Snippet: Paragraph title: Sample preparation for proteomic analysis ... The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad).

    In Vitro:

    Article Title: Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound
    Article Snippet: Eight in vitro samples of fresh right femurs (3.16 ± 0.10 mm in diameter and 34.00 ± 0.30 mm in length) were obtained from eight Wistar rats ( Rattus Norvegicus Albinus ), the samples average weight was 0.54 ± 0.21 g. Rat number 5 did not have epiphysis as it was lost during the bone disarticulation procedure. .. The demineralization protocol consisted of immersing the rat femur samples in a 25-mL solution of Ethylenediaminetetraacetic Acid (EDTA) disodium salt (Sigma-Aldrich®, Missouri, USA) at pH = 8 and 0.376 M concentration for 24 h at 25.0 ± 1.5 °C, and a new solution and new reservoir were used each day .

    Distillation:

    Article Title: Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix
    Article Snippet: Piperidine, calcium hydride, tetrahydrofuran (THF), trimethylsilane (TMS), triethylamine (TEA), tin (II) 2-ethylhexanoate (TOC), acryloyl chloride (AC), acrylic acid, dimethylsulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) disodium salt were purchased from Sigma-Aldrich (St. Louis, MO). .. Dichloromethane (DCM; Acros) was dried by distillation over calcium hydride.

    Produced:

    Article Title: Identification of Extended-Spectrum, AmpC, and Carbapenem- Hydrolyzing ?-Lactamases in Escherichia coli and Klebsiella pneumoniae by Disk Tests
    Article Snippet: The enzymes produced by previously unpublished strains were identified by isoelectric focusing, PCR amplification, cloning, and sequencing, as described previously ( , , , ). .. Plasmids were transferred to porin-deficient K. pneumoniae ), while 3-aminophenylboronic acid (APB) and EDTA disodium salt (EDTA) were obtained from Sigma (St. Louis, MO).

    Concentration Assay:

    Article Title: Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound
    Article Snippet: .. The demineralization protocol consisted of immersing the rat femur samples in a 25-mL solution of Ethylenediaminetetraacetic Acid (EDTA) disodium salt (Sigma-Aldrich®, Missouri, USA) at pH = 8 and 0.376 M concentration for 24 h at 25.0 ± 1.5 °C, and a new solution and new reservoir were used each day . ..

    Article Title: Concentration and time-dependent effect of initial sodium hypochlorite on the ability of QMix and ethylenediaminetetraacetic acid to remove smear layer
    Article Snippet: .. EDTA disodium salt was obtained from Sigma Chemical Co., (St. Louis, MO, USA) and was prepared by diluting the dehydrated salt of EDTA to obtain a concentration of 17% (weight/volume); the pH was adjusted to 7.5 by addition of NaOH. .. Specimen selection and preparation Eighty maxillary central incisors from the patients aged 45–65 years, extracted for periodontal reasons, were stored in 0.1% thymol solution until use.

    Thin Layer Chromatography:

    Article Title: Selection of an optimal macrocyclic chelator improves the imaging of prostate cancer using cobalt-labeled GRPR antagonist RM26
    Article Snippet: Stability was evaluated by instant thin-layer chromatography (ITLC). .. In this experiment, 57 Co-labeled peptides were incubated in the presence of 1000-fold molar excess of EDTA disodium salt (Sigma) for 1 h at RT.

    Lysis:

    Article Title: Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study
    Article Snippet: .. The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad). .. The superfluous salt in the extract was removed by incubation with trichloroacetic acid (TCA)-acetone solution [20% TCA (MERCK, Germany), 20 mmol/L DTT in acetone (MERCK, Germany)] for 4 h at −40°C.

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  • 99
    Millipore nmr buffer
    The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H <t>NMR</t> spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM <t>NaCl,</t> 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .
    Nmr Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nmr buffer/product/Millipore
    Average 99 stars, based on 106 article reviews
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    nmr buffer - by Bioz Stars, 2020-03
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    99
    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore uch buffer
    Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with <t>DLB</t> (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by <t>UCH-L1</t> in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.
    Uch Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore jak2 ip buffer
    Erythropoietin Receptor-Mediated Activation of the <t>JAK2/STAT5,</t> RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).
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    Image Search Results


    The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H NMR spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .

    Journal: PLoS ONE

    Article Title: m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

    doi: 10.1371/journal.pone.0224850

    Figure Lengend Snippet: The m 6 A modification has minor effects on RREIIB structure and dynamics. (A) Secondary structure of RRE2Bm 6A68 , with the residues showing line-broadening with m 6 A, highlighted in red (with Mg 2+ ) and blue (no Mg 2+ ). Comparison of the 1D 1 H NMR spectrum of RREIIB m6A68 with or without Mg 2+ with the methyl peak indicated by arrows. The comparison of 1D imino spectra (B) and 2D [ 1 H, 13 C]-HSQC spectra (C) of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . Resonances exhibiting shifting are indicated using arrows, and those with ambiguous assignments denoted using an asterisk. (D) Normalized resonance intensities in 2D [ 1 H, 13 C]-HSQC spectra of RREIIB m6A68 and RREIIB in the presence (red) and absence (blue) of 3 mM Mg 2+ . A52-C8H8, A52-C2H2 and U56-C6H6 were used as a reference and normalized to 0.1. The sample conditions were 1.2–1.5 mM RREIIB m6A68 or RREIIB in 15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, pH 6.4 with or without 3 mM MgCl 2 .

    Article Snippet: After measuring the concentration, the RNA samples were buffer-exchanged into NMR buffer (15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, with or without 3 mM MgCl2 at pH = 6.4) three times using 3kDa Amicon Ultra centrifugal filters (EMD Millipore).

    Techniques: Modification, Nuclear Magnetic Resonance

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Journal: The American Journal of Pathology

    Article Title: Ubiquitination of ?-Synuclein Is Not Required for Formation of Pathological Inclusions in ?-Synucleinopathies

    doi:

    Figure Lengend Snippet: Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Article Snippet: SDS-soluble fractions from the cingulate cortex of neuropathologically normal (NL) or DLB brains were diluted 40-fold in UCH buffer (50 mmol/L HEPES, pH 7.8, 0.5 mmol/L EDTA, 1 mmol/L dithiothreitol) and then concentrated 40-fold using a MicroconYM-10 (Millipore Corp., Bedford, MA) to remove SDS.

    Techniques: Western Blot, Recombinant, Immunoprecipitation, Isolation, Modification, In Vitro

    Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

    Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Western Blot, Inhibition

    Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Activity Assay, Activation Assay, Immunoprecipitation