ethylenediaminetetraacetic acid  (Millipore)


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    Structured Review

    Millipore ethylenediaminetetraacetic acid
    Effect of decellularisation only on sinew constructs: (a) ultimate tensile stress and (b) maximum modulus values for sinew constructs following decellularisation alone (n = 5/6 in each group). (c) Artificial sinew construct size following treatment with EDTA and SDS as measured by digital imaging. Size refers to the total area of the construct when viewed from above (n = 6 in each group). EDTA: <t>ethylenediaminetetraacetic</t> acid; SDS: sodium dodecyl sulphate. * p
    Ethylenediaminetetraacetic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylenediaminetetraacetic acid/product/Millipore
    Average 75 stars, based on 1 article reviews
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    ethylenediaminetetraacetic acid - by Bioz Stars, 2020-01
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    Images

    1) Product Images from "Combined decellularisation and dehydration improves the mechanical properties of tissue-engineered sinews"

    Article Title: Combined decellularisation and dehydration improves the mechanical properties of tissue-engineered sinews

    Journal: Journal of Tissue Engineering

    doi: 10.1177/2041731414536720

    Effect of decellularisation only on sinew constructs: (a) ultimate tensile stress and (b) maximum modulus values for sinew constructs following decellularisation alone (n = 5/6 in each group). (c) Artificial sinew construct size following treatment with EDTA and SDS as measured by digital imaging. Size refers to the total area of the construct when viewed from above (n = 6 in each group). EDTA: ethylenediaminetetraacetic acid; SDS: sodium dodecyl sulphate. * p
    Figure Legend Snippet: Effect of decellularisation only on sinew constructs: (a) ultimate tensile stress and (b) maximum modulus values for sinew constructs following decellularisation alone (n = 5/6 in each group). (c) Artificial sinew construct size following treatment with EDTA and SDS as measured by digital imaging. Size refers to the total area of the construct when viewed from above (n = 6 in each group). EDTA: ethylenediaminetetraacetic acid; SDS: sodium dodecyl sulphate. * p

    Techniques Used: Construct, Imaging

    2) Product Images from "C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation"

    Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01604

    Purified human endogenous C-reactive protein (eCRP) inhibits BzATP-induced release of interleukin-1β (IL-1β) from U937 cells. Lipopolysaccharide (LPS)-primed (1 µg/ml, 5 h) U937 cells were stimulated with BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt; 100 µM) and IL-1β was measured 30 min later in cell culture supernatants. (A) eCRP dose-dependently inhibited the BzATP-induced IL-1β release, nicotine (Nic; 100 µM) served as a positive control. (B,C) Serum amyloid P (5 µg/ml), human recombinant CRP (rCRP) (5 µg/ml), or low concentrations of free phosphocholine (PC) (1 µM) did not impair IL-1β release, but a combination of rCRP and PC (1 µM) did. (D) The inhibitory effect of eCRP (CRP I; 5 µg/ml) was preserved after ultrafiltration (cutoff 10 kDa; CRP II), but abolished by ultrafiltration in the presence of ethylenediaminetetraacetic acid (1.1 mM; CRP III). PC (1 µM) reconstituted the activity of CRP III, whereas 1 µM PC alone was ineffective. (E) CRP was retained in CRP III and absent from the low molecular weight fraction (LMW). SDS-PAGE followed by staining with Brilliant Blue. (F) The effect of eCRP was reversed by nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (Mec; 100 µM), α-bungarotoxin (α-Bun; 1 µM), strychnine (Stry; 10 µM), ArIB (500 nM), and RgIA4 (200 nM). (G) In experiments using small interfering RNA (siRNA), silencing of the nAChR subunits α7, α9, and α10, but not control siRNA (con) attenuated the inhibition by eCRP. Data are presented as individual data points, bar represents median, whiskers encompass the 25th to 75th percentile. * p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone. # p ≤ 0.05, different LPS-primed cells were stimulated with BzATP and eCRP. Kruskal–Wallis followed by Mann–Whitney rank sum test.
    Figure Legend Snippet: Purified human endogenous C-reactive protein (eCRP) inhibits BzATP-induced release of interleukin-1β (IL-1β) from U937 cells. Lipopolysaccharide (LPS)-primed (1 µg/ml, 5 h) U937 cells were stimulated with BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt; 100 µM) and IL-1β was measured 30 min later in cell culture supernatants. (A) eCRP dose-dependently inhibited the BzATP-induced IL-1β release, nicotine (Nic; 100 µM) served as a positive control. (B,C) Serum amyloid P (5 µg/ml), human recombinant CRP (rCRP) (5 µg/ml), or low concentrations of free phosphocholine (PC) (1 µM) did not impair IL-1β release, but a combination of rCRP and PC (1 µM) did. (D) The inhibitory effect of eCRP (CRP I; 5 µg/ml) was preserved after ultrafiltration (cutoff 10 kDa; CRP II), but abolished by ultrafiltration in the presence of ethylenediaminetetraacetic acid (1.1 mM; CRP III). PC (1 µM) reconstituted the activity of CRP III, whereas 1 µM PC alone was ineffective. (E) CRP was retained in CRP III and absent from the low molecular weight fraction (LMW). SDS-PAGE followed by staining with Brilliant Blue. (F) The effect of eCRP was reversed by nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (Mec; 100 µM), α-bungarotoxin (α-Bun; 1 µM), strychnine (Stry; 10 µM), ArIB (500 nM), and RgIA4 (200 nM). (G) In experiments using small interfering RNA (siRNA), silencing of the nAChR subunits α7, α9, and α10, but not control siRNA (con) attenuated the inhibition by eCRP. Data are presented as individual data points, bar represents median, whiskers encompass the 25th to 75th percentile. * p ≤ 0.05, different from LPS-primed cells stimulated with BzATP alone. # p ≤ 0.05, different LPS-primed cells were stimulated with BzATP and eCRP. Kruskal–Wallis followed by Mann–Whitney rank sum test.

    Techniques Used: Purification, Cell Culture, Positive Control, Recombinant, Activity Assay, Molecular Weight, SDS Page, Staining, Small Interfering RNA, Inhibition, MANN-WHITNEY

    3) Product Images from "Anti-Escherichia coli O157:H7 Properties of Purple Prairie Clover and Sainfoin Condensed Tannins"

    Article Title: Anti-Escherichia coli O157:H7 Properties of Purple Prairie Clover and Sainfoin Condensed Tannins

    Journal: Molecules

    doi: 10.3390/molecules18022183

    Quenching of 1- N -phenylnaphthylamine (NPN) fluorescence by addition of condensed tannins (0, 50 and 200 µg/mL; a ) from purple prairie clover (PPC; Dalea purpurea Vent.) and sainfoin (SF; Onobrychis viciifolia ), in comparison with addition of ethylenediaminetetraacetic acid (EDTA; b ), to Escherichia coli (ATCC 25922) suspension prepared from non-exposed bacteria. Bars indicate standard error.
    Figure Legend Snippet: Quenching of 1- N -phenylnaphthylamine (NPN) fluorescence by addition of condensed tannins (0, 50 and 200 µg/mL; a ) from purple prairie clover (PPC; Dalea purpurea Vent.) and sainfoin (SF; Onobrychis viciifolia ), in comparison with addition of ethylenediaminetetraacetic acid (EDTA; b ), to Escherichia coli (ATCC 25922) suspension prepared from non-exposed bacteria. Bars indicate standard error.

    Techniques Used: Fluorescence

    1- N -Phenylnaphthylamine (NPN) fluorescence of Escherichia coli (ATCC25922) pre-cultured with ethylenediaminetetraacetic acid (EDTA; 0.05µmol/mL) or condensed tannins (10 µg/mL) from purple prairie clover (PPC; Dalea purpurea Vent.) and sainfoin (SF; Onobrychis viciifolia ). Bars indicate standard error.
    Figure Legend Snippet: 1- N -Phenylnaphthylamine (NPN) fluorescence of Escherichia coli (ATCC25922) pre-cultured with ethylenediaminetetraacetic acid (EDTA; 0.05µmol/mL) or condensed tannins (10 µg/mL) from purple prairie clover (PPC; Dalea purpurea Vent.) and sainfoin (SF; Onobrychis viciifolia ). Bars indicate standard error.

    Techniques Used: Fluorescence, Cell Culture

    4) Product Images from "Isolation of a Klebsiella pneumoniae Isolate of Sequence Type 258 Producing KPC-2 Carbapenemase in Korea"

    Article Title: Isolation of a Klebsiella pneumoniae Isolate of Sequence Type 258 Producing KPC-2 Carbapenemase in Korea

    Journal: The Korean Journal of Laboratory Medicine

    doi: 10.3343/kjlm.2011.31.4.298

    Results obtained with a Modified Hodge test (A) and carbapenemase inhibition test (B) for carbapenem-resistant Klebsiella pneumoniae isolate (KPN 1010). Abbreviations: MEM, meropenem; APB, aminophenylboronic acid; CLX, cloxacillin; EDTA, ethylenediaminetetraacetic acid; DPA, dipicolinic acid.
    Figure Legend Snippet: Results obtained with a Modified Hodge test (A) and carbapenemase inhibition test (B) for carbapenem-resistant Klebsiella pneumoniae isolate (KPN 1010). Abbreviations: MEM, meropenem; APB, aminophenylboronic acid; CLX, cloxacillin; EDTA, ethylenediaminetetraacetic acid; DPA, dipicolinic acid.

    Techniques Used: Modification, Inhibition

    Related Articles

    Centrifugation:

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The resulting pCW plasmid was transformed into E.Coli (BL21) and cultured overnight. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma). .. After being centrifuged (16,000 x g , 10 min) at 4°C, the supernatant was applied to a 2',5'-ADP-Sepharose 4B column pre-equilibrated in buffer A.

    Co-Culture Assay:

    Article Title: Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression
    Article Snippet: Paragraph title: 4.3.1. Individual and Co-Culture Experiments ... Afterwards, cells were stimulated with 3.2 mg EDTA (Sigma Aldrich, Darmstadt, Germany), 10 µL HMWH (=50 IU; Ratiopharm, Ulm, Germany) or LMWH (Clexane; Sanofi-Aventis, Frankfurt, Germany or Fragmin; Pfizer, Berlin, Germany), or 220 µL citrate (Sarstedt) per well and harvested after 0, 4, 6, and 24 h for the analysis of MMP-9 mRNA expression.

    Incubation:

    Article Title: A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture
    Article Snippet: After thawing and sequential washing in PBS for 10–30 min at room temperature to remove DMSO or glycerol, HAM was de-epithelialized using different methods for comparison as follows: EDTA treatment. .. HAM was incubated in 0.02% EDTA (Sigma-Aldrich, cat# E-5134) in calcium-magnesium free PBS at 37°C for one hour to loosen amniotic epithelial cells, and then transferred into PBS. .. Treatment of membranes was followed by immediately washing twice for 15 min in PBS to remove cellular debris.

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: The resultant mixture (10 μM) was amplified by incubating at 30 °C for 16 h in a solution containing 2.5 U/μL phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM (NH4 )2 SO4 , 4 mM DTT, 200 μg/ml BSA, and 2.5 mM dNTP (Invitrogen, Carlsbad, CA, USA). .. Polypodna production by restriction digestion The highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product. .. After purification by size-exclusion chromatography, the resultant large molecular weight DNA was digested with 0.1 U/μL TspRI (New England Biolabs) in solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg/ml BSA.

    Expressing:

    Article Title: Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression
    Article Snippet: In the initial experiments, 2 × 106 THP-1, Jurkat, or HT cells/well were starved in individual cultures overnight. .. Afterwards, cells were stimulated with 3.2 mg EDTA (Sigma Aldrich, Darmstadt, Germany), 10 µL HMWH (=50 IU; Ratiopharm, Ulm, Germany) or LMWH (Clexane; Sanofi-Aventis, Frankfurt, Germany or Fragmin; Pfizer, Berlin, Germany), or 220 µL citrate (Sarstedt) per well and harvested after 0, 4, 6, and 24 h for the analysis of MMP-9 mRNA expression. .. In double co-culture experiments, THP-1 and Jurkat, THP-1 and HT, or Jurkat and HT cells were cultivated together (in total 2 × 106 cells/well, i.e., 1 × 106 cells per cell line).

    Transformation Assay:

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The resulting pCW plasmid was transformed into E.Coli (BL21) and cultured overnight. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma).

    High Performance Liquid Chromatography:

    Article Title: Membrane Interacting Regions of Dengue Virus NS2A Protein
    Article Snippet: The composition of the synthetic endoplasmic reticulum was EPC/CL/BPI/TPE/BPS/EPA/SM/Chol in the following proportion 59:0.37:7.7:18:3.1:1.2:3.4:7.8. , According to the manufacturer, the liver lipid extract contains 42% phosphatidylcholine, 22% phosphatidylethanolamine, 7% Chol, 8% phosphatidylinositol, 1% lysophosphatidylinositol, and 21% miscellaneous lipids including neutral ones. .. 5-Carboxyfluorescein (CF, > 95% by HPLC), deuterium oxide (99.9% by atom), Triton X-100, EDTA, and HEPES were acquired from Sigma-Aldrich (Madrid, Spain). .. 1,6-Diphenyl-1,3,5-hexatriene (DPH) and N -(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn -glycero-3-phosphoethanolamine (fluorescein DHPE or FPE) were acquired from Molecular Probes (Eugene, OR, USA).

    Electron Microscopy:

    Article Title: Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts
    Article Snippet: Paragraph title: Electron Microscopy ... 18 μl samples of prey ghosts were treated with 2 μl deionised water (control) 1%SDS (+SDS) or 1 mg ml−1 chicken’s egg lysozyme (Sigma) 100 mM EDTA (+ lysozyme) for 1 hour.

    Flow Cytometry:

    Article Title: Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding
    Article Snippet: For the EDTA-DET treatment, rat livers were perfused with 2mM EDTA (Sigma, UK) for 15 minutes followed by dH2 O for 36 hours at 4°C. .. For the EDTA-DET treatment, rat livers were perfused with 2mM EDTA (Sigma, UK) for 15 minutes followed by dH2 O for 36 hours at 4°C.

    Protease Inhibitor:

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The resulting pCW plasmid was transformed into E.Coli (BL21) and cultured overnight. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma). .. After being centrifuged (16,000 x g , 10 min) at 4°C, the supernatant was applied to a 2',5'-ADP-Sepharose 4B column pre-equilibrated in buffer A.

    Cell Culture:

    Article Title: Anti-Escherichia coli O157:H7 Properties of Purple Prairie Clover and Sainfoin Condensed Tannins
    Article Snippet: The sub-lethal concentration of 10 µg/mL used in the pre-incubation was based on the MIC of PPC tannin (25–50% of MIC). .. In addition, bacteria was also aerobically cultured in two flasks with M9 media containing 0.05 µmol/mL of ethylenediaminetetraacetic acid (EDTA; Sigma) in the same manner as above for comparison since EDTA is a commonly used membrane permeabilizer. .. The bacterial cells suspensions that were used for the OM permeability were prepared in the same manner as described in , but using phosphate buffer.

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The resulting pCW plasmid was transformed into E.Coli (BL21) and cultured overnight. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma).

    Hemagglutination Assay:

    Article Title: Metal chelator combined with permeability enhancer ameliorates oxidative stress-associated neurodegeneration in rat eyes with elevated intraocular pressure
    Article Snippet: EDTA-MSM was applied topically to the eyes of rats to ameliorate the sequelae of oxidative stress and neurodegeneration induced by chronically elevated intraocular pressure. .. Hyaluronic acid (HA), kanamycin solution, EDTA, and MSM were purchased from Sigma Aldrich Inc. (St. Louis, MO). .. Phosphate-buffered saline (PBS) solution, dimethyl sulfoxide (DMSO), and staining reagents were purchased from Invitrogen Corporation (Carlsbad, CA).

    other:

    Article Title: The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease
    Article Snippet: E-64, DTT, l -cysteine, GSH (reduced glutathione), EDTA and ovalbumin were obtained from Sigma-Aldrich (Sydney, Australia).

    Article Title: Mechanistic insight into cadmium-induced inactivation of the Bloom protein
    Article Snippet: CdCl2 , ZnCl2 , EDTA, DTT (dithiothreitol), NEM (N-Ethylmaleimide), ATP and Triton X-100 were purchased from Sigma.

    Article Title: An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane
    Article Snippet: SDS and EDTA, purchased from Sigma-Aldrich, were prepared as filter sterilized stock solutions of 10% SDS and 50 mM EDTA (pH 7.5).

    Imaging:

    Article Title: Functional interaction of human Ssu72 with RNA polymerase II complexes
    Article Snippet: If RNA was to be isolated, elongation complexes were stopped with stop solution containing 100 mM Tris pH 7.6, 0.2 mg/ml Torula Yeast RNA, 20 mM EDTA, and 1% Sarkosyl (Sigma, R6625). .. If RNA was to be isolated, elongation complexes were stopped with stop solution containing 100 mM Tris pH 7.6, 0.2 mg/ml Torula Yeast RNA, 20 mM EDTA, and 1% Sarkosyl (Sigma, R6625).

    Sequencing:

    Article Title: Membrane Interacting Regions of Dengue Virus NS2A Protein
    Article Snippet: The peptide dens25 corresponding to the sequence KHAILLVAVSFVTLITGNMSFRDLGR from Dengue Virus Type 2 NGC NS2A protein (with N-terminal acetylation and C-terminal acetylation) and a purity greater than 95% was obtained from Genemed Synthesis, San Antonio, TX, USA. .. 5-Carboxyfluorescein (CF, > 95% by HPLC), deuterium oxide (99.9% by atom), Triton X-100, EDTA, and HEPES were acquired from Sigma-Aldrich (Madrid, Spain).

    Molecular Weight:

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: Polypodna production by restriction digestion The highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product. .. After purification by size-exclusion chromatography, the resultant large molecular weight DNA was digested with 0.1 U/μL TspRI (New England Biolabs) in solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg/ml BSA.

    Fluorescence:

    Article Title: Oxidized Phospholipids as Potential Novel Drug Targets
    Article Snippet: HPD, CPZ, DOX, NaCl, Hepes, EDTA, calf thymus DNA, and polyphosphate were from Sigma (St. Louis, MO) and CaCl2 and DMSO from Merck (Darmstadt, Germany). .. The purity of the lipids was checked by thin layer chromatography on silicic acid-coated plates (Merck), using chloroform/methanol/water/ammonia (65:20:2:2, v/v) as the eluent.

    Isolation:

    Article Title: Functional interaction of human Ssu72 with RNA polymerase II complexes
    Article Snippet: If complexes were to be isolated, see EC-EMSA protocol. .. If RNA was to be isolated, elongation complexes were stopped with stop solution containing 100 mM Tris pH 7.6, 0.2 mg/ml Torula Yeast RNA, 20 mM EDTA, and 1% Sarkosyl (Sigma, R6625). .. The RNA was then isolated by phenol extraction and precipitated by addition of 3 volumes of 95% ethanol containing 0.5 M ammonium acetate.

    Size-exclusion Chromatography:

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: Polypodna production by restriction digestion The highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product. .. After purification by size-exclusion chromatography, the resultant large molecular weight DNA was digested with 0.1 U/μL TspRI (New England Biolabs) in solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg/ml BSA.

    Microscopy:

    Article Title: Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts
    Article Snippet: 18 μl samples of prey ghosts were treated with 2 μl deionised water (control) 1%SDS (+SDS) or 1 mg ml−1 chicken’s egg lysozyme (Sigma) 100 mM EDTA (+ lysozyme) for 1 hour. .. 15 μl samples were placed on Veco copper grids, 200 mesh for 2–5 minutes, washed with PBS for 1–2 minutes and stained with 15 μl 2% phosphotungstic acid (PTA) solution pH 7.0 for 30 s to 2 minutes.

    Purification:

    Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation
    Article Snippet: dUMP, dTMP, 5F-dUMP, sodium dithionite, EDTA, formaldehyde, and l -cysteine were purchased from Sigma Chemical Co. dUMPS was from Axxora, LLC. .. dUMP, dTMP, 5F-dUMP, sodium dithionite, EDTA, formaldehyde, and l -cysteine were purchased from Sigma Chemical Co. dUMPS was from Axxora, LLC.

    Article Title: Formation of Three-Dimensional Structures in Supported Lipid Bilayers
    Article Snippet: Chloroform stock solutions of 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn -glycero-3-phosphate (DOPA), and 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]- sn -glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids and used without further purification. .. 2-( N -Morpholino)ethanesulfonic acid hydrate (MES hydrate) and EDTA were purchased from Sigma Chemical (St. Louis, MO).

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: Polypodna production by restriction digestion The highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product. .. After purification by size-exclusion chromatography, the resultant large molecular weight DNA was digested with 0.1 U/μL TspRI (New England Biolabs) in solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg/ml BSA.

    Article Title: Effect of Ions on the Organization of Phosphatidylcholine/Phosphatidic Acid Bilayers
    Article Snippet: Chloroform stock solutions of DOPC, DOPA, and 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]- sn -glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. .. 2-( N -Morpholino)ethanesulfonic acid hydrate (MES hydrate), phosphate-buffered saline (PBS) tablets, and EDTA were purchased from Sigma Chemical (Poole, Dorset, UK).

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: Paragraph title: ENOS Purification ... After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma).

    Polymerase Chain Reaction:

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The PCR products were digested with EcoRI and XbaI and the product was subcloned into pCW plasmid [ , – ]. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma).

    Plasmid Preparation:

    Article Title: The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain
    Article Snippet: The resulting pCW plasmid was transformed into E.Coli (BL21) and cultured overnight. .. After harvesting the E.Coli by centrifugation, the bacteria pellet was homogenized in buffer A, which contains 50 mM Tris·HCl, pH 7.6, 0.1 mM EDTA, 150mM NaCl, 0.1mM DTT, 10% glycerol and protease inhibitor cocktail solution (Sigma).

    In Vitro:

    Article Title: Functional interaction of human Ssu72 with RNA polymerase II complexes
    Article Snippet: Paragraph title: In vitro transcription assay ... If RNA was to be isolated, elongation complexes were stopped with stop solution containing 100 mM Tris pH 7.6, 0.2 mg/ml Torula Yeast RNA, 20 mM EDTA, and 1% Sarkosyl (Sigma, R6625).

    Concentration Assay:

    Article Title: Anti-Escherichia coli O157:H7 Properties of Purple Prairie Clover and Sainfoin Condensed Tannins
    Article Snippet: The sub-lethal concentration of 10 µg/mL used in the pre-incubation was based on the MIC of PPC tannin (25–50% of MIC). .. In addition, bacteria was also aerobically cultured in two flasks with M9 media containing 0.05 µmol/mL of ethylenediaminetetraacetic acid (EDTA; Sigma) in the same manner as above for comparison since EDTA is a commonly used membrane permeabilizer.

    Article Title: Oxidized Phospholipids as Potential Novel Drug Targets
    Article Snippet: HPD, CPZ, DOX, NaCl, Hepes, EDTA, calf thymus DNA, and polyphosphate were from Sigma (St. Louis, MO) and CaCl2 and DMSO from Merck (Darmstadt, Germany). .. Examination of the plates after iodine staining or, when appropriate, for fluorescence upon UV illumination revealed no impurities.

    Article Title: Activity of Amphotericin B and Anidulafungin Combined with Rifampicin, Clarithromycin, Ethylenediaminetetraacetic Acid, N-Acetylcysteine, and Farnesol against Candida tropicalis Biofilms
    Article Snippet: AMB, EDTA, NAC, and FAR were provided by Sigma-Aldrich (Madrid, Spain) and dissolved in dimethyl sulfoxide (AMB, FAR) and water (EDTA, NAC). .. AMB, EDTA, NAC, and FAR were provided by Sigma-Aldrich (Madrid, Spain) and dissolved in dimethyl sulfoxide (AMB, FAR) and water (EDTA, NAC).

    Staining:

    Article Title: Oxidized Phospholipids as Potential Novel Drug Targets
    Article Snippet: HPD, CPZ, DOX, NaCl, Hepes, EDTA, calf thymus DNA, and polyphosphate were from Sigma (St. Louis, MO) and CaCl2 and DMSO from Merck (Darmstadt, Germany). .. The purity of the lipids was checked by thin layer chromatography on silicic acid-coated plates (Merck), using chloroform/methanol/water/ammonia (65:20:2:2, v/v) as the eluent.

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    Millipore pbs edta
    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as <t>PBS-EDTA,</t> to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
    Pbs Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs edta/product/Millipore
    Average 93 stars, based on 4 article reviews
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    78
    Millipore edta free buffer
    Procedures for <t>EDTA</t> removal from protein samples and detection of EDTA. (A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or <t>vimentin</t> were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl 2 . A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn 2+ -EDTA chelate are clearly visible.
    Edta Free Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free buffer/product/Millipore
    Average 78 stars, based on 2 article reviews
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    79
    Millipore caspase reaction buffer
    Cleavage of SREBP-1 in ethanol-exposed cells treated with TNF is insensitive to inhibition by cholesterol but is dependent on activation of <t>caspase-4</t> or -12. A , McA-RH 7777 and HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. Where indicated, TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells were then incubated for a further 24 h. The cells were then harvested and assessed for cleavage of SREBP-1 by Western blotting as described under “Experimental Procedures.” B , McA-RH 7777 or HepG2 cells were plated into 6-well 9.3-cm2 plates at 1.0 × 106 cells. Cells were exposed to 25 m m ethanol in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells incubated for a further 24 h. Where indicated, 10 μ m caspase-4 or -12 inhibitors, LEVD-FMK or ATAD-FMK, respectively, were added in tandem with the TNF. Caspase-4 or -12 activity was measured as detailed under “Experimental Procedures.” C , first lanes , McA-RH 7777 or HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence of 10 μ m of the caspase-4 or -12 inhibitors. In the second lane , the cells were first exposed to 25 m m ethanol in the presence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the medium after 24 h of ethanol exposure and the cells incubated for a further 24 h, whereas, in the third lane , 10 μ m caspase-4 or -12 inhibitors were added in tandem with TNF.
    Caspase Reaction 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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    Image Search Results


    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Article Snippet: All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

    Techniques: Concentration Assay, Buffer Exchange

    (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Article Snippet: All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

    Techniques: Injection, Flow Cytometry, Size-exclusion Chromatography

    (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Article Snippet: All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

    Techniques: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

    Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Article Snippet: All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

    Techniques: Solubility, Centrifugation, Concentration Assay, Standard Deviation

    Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl 2 . A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn 2+ -EDTA chelate are clearly visible.

    Journal: PLoS ONE

    Article Title: Drawbacks of Dialysis Procedures for Removal of EDTA

    doi: 10.1371/journal.pone.0169843

    Figure Lengend Snippet: Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl 2 . A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn 2+ -EDTA chelate are clearly visible.

    Article Snippet: For ultrafiltration, BSA or vimentin samples (250 μl) containing 1 mM EDTA were diluted 10-fold with EDTA-free buffer, applied to Millipore Amicon Ultra filter units (10 K pore size) and centrifuged at 3000xg for 15 min at 16°C, which concentrated the samples down to their original volume.

    Techniques: Purification, Filtration, Competitive Binding Assay, Nuclear Magnetic Resonance, Proton NMR, Concentration Assay

    Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz 1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl 2 . Signals for the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm (2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn 2+ , the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl 2 . Lower panel: Amount of EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the absorbance at 492 nm after incubation with PAR and ZnCl 2 , using the calibration curve. Initial EDTA concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays.

    Journal: PLoS ONE

    Article Title: Drawbacks of Dialysis Procedures for Removal of EDTA

    doi: 10.1371/journal.pone.0169843

    Figure Lengend Snippet: Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz 1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl 2 . Signals for the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm (2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn 2+ , the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl 2 . Lower panel: Amount of EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the absorbance at 492 nm after incubation with PAR and ZnCl 2 , using the calibration curve. Initial EDTA concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays.

    Article Snippet: For ultrafiltration, BSA or vimentin samples (250 μl) containing 1 mM EDTA were diluted 10-fold with EDTA-free buffer, applied to Millipore Amicon Ultra filter units (10 K pore size) and centrifuged at 3000xg for 15 min at 16°C, which concentrated the samples down to their original volume.

    Techniques: Quantitation Assay, Nuclear Magnetic Resonance, Proton NMR, Concentration Assay, Incubation

    Cleavage of SREBP-1 in ethanol-exposed cells treated with TNF is insensitive to inhibition by cholesterol but is dependent on activation of caspase-4 or -12. A , McA-RH 7777 and HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. Where indicated, TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells were then incubated for a further 24 h. The cells were then harvested and assessed for cleavage of SREBP-1 by Western blotting as described under “Experimental Procedures.” B , McA-RH 7777 or HepG2 cells were plated into 6-well 9.3-cm2 plates at 1.0 × 106 cells. Cells were exposed to 25 m m ethanol in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells incubated for a further 24 h. Where indicated, 10 μ m caspase-4 or -12 inhibitors, LEVD-FMK or ATAD-FMK, respectively, were added in tandem with the TNF. Caspase-4 or -12 activity was measured as detailed under “Experimental Procedures.” C , first lanes , McA-RH 7777 or HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence of 10 μ m of the caspase-4 or -12 inhibitors. In the second lane , the cells were first exposed to 25 m m ethanol in the presence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the medium after 24 h of ethanol exposure and the cells incubated for a further 24 h, whereas, in the third lane , 10 μ m caspase-4 or -12 inhibitors were added in tandem with TNF.

    Journal:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive

    doi: 10.1074/jbc.M800237200

    Figure Lengend Snippet: Cleavage of SREBP-1 in ethanol-exposed cells treated with TNF is insensitive to inhibition by cholesterol but is dependent on activation of caspase-4 or -12. A , McA-RH 7777 and HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. Where indicated, TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells were then incubated for a further 24 h. The cells were then harvested and assessed for cleavage of SREBP-1 by Western blotting as described under “Experimental Procedures.” B , McA-RH 7777 or HepG2 cells were plated into 6-well 9.3-cm2 plates at 1.0 × 106 cells. Cells were exposed to 25 m m ethanol in the presence or absence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the cell culture medium after 24 h of ethanol exposure and the cells incubated for a further 24 h. Where indicated, 10 μ m caspase-4 or -12 inhibitors, LEVD-FMK or ATAD-FMK, respectively, were added in tandem with the TNF. Caspase-4 or -12 activity was measured as detailed under “Experimental Procedures.” C , first lanes , McA-RH 7777 or HepG2 cells were exposed to 25 m m ethanol for 48 h in the presence of 10 μ m of the caspase-4 or -12 inhibitors. In the second lane , the cells were first exposed to 25 m m ethanol in the presence of cholesterol and 25-hydroxycholesterol at concentrations of 10 and 1 μg/ml, respectively. TNF (1 ng/ml) was added to the medium after 24 h of ethanol exposure and the cells incubated for a further 24 h, whereas, in the third lane , 10 μ m caspase-4 or -12 inhibitors were added in tandem with TNF.

    Article Snippet: The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Techniques: Inhibition, Activation Assay, Cell Culture, Incubation, Western Blot, Activity Assay

    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Journal:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive

    doi: 10.1074/jbc.M800237200

    Figure Lengend Snippet: TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Article Snippet: The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Techniques: Activation Assay