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Fisher Scientific ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic Acid, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ethylenediaminetetraacetic acid - by Bioz Stars, 2020-03
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Clone Assay:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A PCR with Fox1-specific primers (Table ) resulted in the amplification of a 761-bp product (nucleotides 1476 to 2237; amino acids His394 to Val646) that was digested with Bam HI, cloned in frame into the 3′ terminus of the TrxA-encoding sequence of the expression vector pTrxFus, and introduced into E. coli for tryptophan-inducible expression (Invitrogen). .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Centrifugation:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling). ..

Amplification:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A PCR with Fox1-specific primers (Table ) resulted in the amplification of a 761-bp product (nucleotides 1476 to 2237; amino acids His394 to Val646) that was digested with Bam HI, cloned in frame into the 3′ terminus of the TrxA-encoding sequence of the expression vector pTrxFus, and introduced into E. coli for tryptophan-inducible expression (Invitrogen). .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Blocking Assay:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Staining was performed exactly as described (DIG Wash & Block Buffer Set, DIG Staining Solution, Roche) by incubating sections with wash buffer, blocking reagent, then alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:250, 1 h in a humidified chamber at room temperature), washing twice and developing the color overnight at room temperature in the presence of 1 mM levamisole (Sigma, St. Louis, MO) to inhibit endogenous phosphatase activity. .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

Electrophoresis:

Article Title: Barnacle cement: a polymerization model based on evolutionary concepts
Article Snippet: Paragraph title: Electrophoresis of cement with Ca2+ chelators ... EGTA (Sigma-Aldrich no. E4378) and EDTA (Fisher Chemical, no. BP118) were used.

Incubation:

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem). .. Membranes were subsequently incubated with appropriate anti-rabbit or anti-mouse IRDye (680 or 800)-labeled antibodies and detected using the LiCOR Biosciences Odyssey system.

Activity Assay:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Staining was performed exactly as described (DIG Wash & Block Buffer Set, DIG Staining Solution, Roche) by incubating sections with wash buffer, blocking reagent, then alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:250, 1 h in a humidified chamber at room temperature), washing twice and developing the color overnight at room temperature in the presence of 1 mM levamisole (Sigma, St. Louis, MO) to inhibit endogenous phosphatase activity. .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

Infection:

Article Title: Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice
Article Snippet: ELISA of proteins in cerebral tissue Brain tissue extracts were obtained from control and infected mice and stored at -20°C. .. Thereafter, the brain tissue was homogenized in an extraction solution (100 mg of tissue per 1 mL of extraction solution) containing 0.4 mol/L NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 mmol/L phenylmethyl sulfonil fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 20 KI aprotinin, using Ultra-Turrax (Fisher Scientific, Pittsburgh, PA).

Expressing:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: E. coli cells expressing the fusion protein were cultured and harvested as described in the manufacturer's protocols. .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: Paragraph title: Detection of β-Arrestin Expression and Phospholamban Phosphorylation. ... Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem).

BIA-KA:

Article Title: Aging Is Not Associated with Proteasome Impairment in UPS Reporter Mice
Article Snippet: Immunoblotting procedures Each of the 6 sub-dissected regions from the left hemisphere and the spinal cord were weighed and homogenized in 10× volume of homogenate buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitors I and II [Fisher Scientific]). .. Immunoblotting procedures Each of the 6 sub-dissected regions from the left hemisphere and the spinal cord were weighed and homogenized in 10× volume of homogenate buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitors I and II [Fisher Scientific]).

Hybridization:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Prehybridization and hybridization were carried out in 50% formamide, essentially as described [ ], with 400 ng/mL RNA probes under high stringency conditions to minimize cross-reactivity between the target genes and closely related Cyp family members. .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

High Performance Liquid Chromatography:

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles
Article Snippet: All buffers (HEPES, MES, CHES), as well as EDTA and NaCl, were of enzyme grade purity (Fisher Scientific, Chicago, IL). .. Chloroform and methanol, which were used to prepare lipid stock solutions, were ACS grade, whereas the water used for buffer preparation was HPLC grade (all Fisher Scientific, Chicago, IL).

Flow Cytometry:

Article Title: Antioxidant Chemistry of Graphene-Based Materials and its Role in Oxidation Protection Technology
Article Snippet: RGO was produced by heating multilayer GO flakes at 250 C for 30 min in nitrogen flow. .. Hydrogen peroxide (H2 O2 ), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA).

Protease Inhibitor:

Article Title: Aging Is Not Associated with Proteasome Impairment in UPS Reporter Mice
Article Snippet: .. Immunoblotting procedures Each of the 6 sub-dissected regions from the left hemisphere and the spinal cord were weighed and homogenized in 10× volume of homogenate buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitors I and II [Fisher Scientific]). .. Following sonication, samples were centrifuged at 16,000 g for 15 minutes, and a BCA protein assay (Thermo Scientific) performed on the supernatant.

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: .. Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem). .. Lysates were run on 8% SDS/PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2-mm pore size membranes (Millipore).

Cell Culture:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: E. coli cells expressing the fusion protein were cultured and harvested as described in the manufacturer's protocols. .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

In Situ Hybridization:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Paragraph title: In situ hybridization histochemistry ... Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

Generated:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A thioredoxin (TrxA)-Fox1 fusion protein was generated for the production of Fox1 antiserum. .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

other:

Article Title: Detection of Carbapenemase Production in Gram-negative Bacteria
Article Snippet: MATERIALS AND REAGENTS REQUIRED FOR PHENOTYPIC METHODS Petri-plates (Hi-Media, Mumbai/Similar standards) Antibiotic discs (Becton Dickson, Franklin lakes, New Jersey, USA/Similar standards) Incubator (Widsons Scientific Works, New Delhi, India) E-test strips (Biomerieux, France) Swab sticks Normal saline (Biomerieux, France) 0.5 M EDTA (Fischer Scientific, Hampton, New Hamptonshire, USA, Catalogue no-12635) 2-mercaptopropionic acid (3 μl) (Hi-Media, Mumbai, India-RM4725-100G) Micropipettes (20-200 μl, 0.5-10 μl) – (Eppendorf, Hamburg, Germany) Phenyl boronic acid (Sigma Aldrich, St. Louis, MO, USA-78181-10G) Dimethyl Sulfoxide (20 mg of phenyl boronic acid is dissolved in 1 ml of DMSO (Ameresco, Massachusetts, USA Lot no-0031C474) 0.5 McFarland standard.

Article Title: Development of a β-Lactoglobulin Sensor Based on SPR for Milk Allergens Detection
Article Snippet: Sodium chloride (NaCl), sodium hydroxide (NaOH), 2-(N -morpholino)ethanesulfonic acid (MES), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), HBS-EP = 10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween-20) were supplied by Fischer Scientific UK (Loughborough, UK).

Article Title: Removal of matrix-bound zoledronate prevents post-extraction osteonecrosis of the jaw by rescuing osteoclast function
Article Snippet: To investigate the effect of chelation-induced removal of Zol on osteoclast differentiation, we repeated the osteoclast differentiation experiment on the osteoassay plate as described above, with addition of EDTA 17% (Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate, Fisher Scientific, Inc., Aiken Rd, Asheville, NC, USA) for 30 minutes after the 24-hour Zol treatment.

Article Title: Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site
Article Snippet: Monobasic and dibasic potassium phosphate, acetonitrile (Optima grade), formic acid, sodium hydroxide, yeast extract, peptone, dextrose (glucose), EDTA, sodium chloride, and sodium acetate were obtained from Fischer Scientific.

Sequencing:

Article Title: Selective disulfide reduction for labeling and enhancement of Fab antibody fragments
Article Snippet: .. Tris base, NaCl, EDTA, and other buffer components, as well as sequencing grade concentrated formic acid, were from Fisher Scientific. .. Fab fragment was generated and purified from the h2E2 anti-cocaine mAb as described previously [ ], using Endoproteinase Lys-C for proteolysis (rather than papain).

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A PCR with Fox1-specific primers (Table ) resulted in the amplification of a 761-bp product (nucleotides 1476 to 2237; amino acids His394 to Val646) that was digested with Bam HI, cloned in frame into the 3′ terminus of the TrxA-encoding sequence of the expression vector pTrxFus, and introduced into E. coli for tryptophan-inducible expression (Invitrogen). .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Sonication:

Article Title: Aging Is Not Associated with Proteasome Impairment in UPS Reporter Mice
Article Snippet: Immunoblotting procedures Each of the 6 sub-dissected regions from the left hemisphere and the spinal cord were weighed and homogenized in 10× volume of homogenate buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitors I and II [Fisher Scientific]). .. Immunoblotting procedures Each of the 6 sub-dissected regions from the left hemisphere and the spinal cord were weighed and homogenized in 10× volume of homogenate buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitors I and II [Fisher Scientific]).

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling). ..

Nucleic Acid Electrophoresis:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling). .. The inclusion bodies were collected by centrifugation (5 min at 14,000 × g , 4°C), washed three times with cold TEN, solubilized with sample buffer (50 mM Tris-HCl [pH 6.8], 5% [vol/vol] 2-mercaptoethanol, 2% [wt/vol] sodium dodecyl sulfate [SDS], 0.1% [wt/vol] bromophenol blue, 10% [wt/vol] glycerol), and subjected to preparative SDS-polyacrylamide gel electrophoresis (12% acrylamide).

Isolation:

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: .. Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem). .. Lysates were run on 8% SDS/PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2-mm pore size membranes (Millipore).

Microscopy:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy. ..

Mouse Assay:

Article Title: Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice
Article Snippet: ELISA of proteins in cerebral tissue Brain tissue extracts were obtained from control and infected mice and stored at -20°C. .. Thereafter, the brain tissue was homogenized in an extraction solution (100 mg of tissue per 1 mL of extraction solution) containing 0.4 mol/L NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 mmol/L phenylmethyl sulfonil fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 20 KI aprotinin, using Ultra-Turrax (Fisher Scientific, Pittsburgh, PA).

Polymerase Chain Reaction:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A PCR with Fox1-specific primers (Table ) resulted in the amplification of a 761-bp product (nucleotides 1476 to 2237; amino acids His394 to Val646) that was digested with Bam HI, cloned in frame into the 3′ terminus of the TrxA-encoding sequence of the expression vector pTrxFus, and introduced into E. coli for tryptophan-inducible expression (Invitrogen). .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Labeling:

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles
Article Snippet: Fluorescently labeled D(+)- sn -1- O -[1-[6′-[6-[((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza- s -indacene-3-yl) phenoxy) acetyl) amino]hexanoyl]amino]hexanoyl]-2-hexanoylglyceryl D- myo- phosphatidylinositol 3-phosphate (BODIPY-PI-3P; excitation = 589 nm; emission = 617 nm) as well as the corresponding BODIPY-PI-4P and -PI-5P were obtained from Molecular Probes (Eugene, OR; purity > 95%). .. All buffers (HEPES, MES, CHES), as well as EDTA and NaCl, were of enzyme grade purity (Fisher Scientific, Chicago, IL).

Staining:

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Staining was performed exactly as described (DIG Wash & Block Buffer Set, DIG Staining Solution, Roche) by incubating sections with wash buffer, blocking reagent, then alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (1:250, 1 h in a humidified chamber at room temperature), washing twice and developing the color overnight at room temperature in the presence of 1 mM levamisole (Sigma, St. Louis, MO) to inhibit endogenous phosphatase activity. .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling). .. The region of the gel containing the fusion protein (visualized by zinc-imidazole staining) was excised and used directly for antiserum production in rabbits (service provided by Covance Research Products, Denver, Pa.).

SDS Page:

Article Title: Barnacle cement: a polymerization model based on evolutionary concepts
Article Snippet: The effect of divalent cation chelators on cement polymerization was tested using SDS-PAGE (under reducing conditions), as described above for soybean trypsin inhibitor. .. EGTA (Sigma-Aldrich no. E4378) and EDTA (Fisher Chemical, no. BP118) were used.

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem). .. Lysates were run on 8% SDS/PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2-mm pore size membranes (Millipore).

Plasmid Preparation:

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii
Article Snippet: A PCR with Fox1-specific primers (Table ) resulted in the amplification of a 761-bp product (nucleotides 1476 to 2237; amino acids His394 to Val646) that was digested with Bam HI, cloned in frame into the 3′ terminus of the TrxA-encoding sequence of the expression vector pTrxFus, and introduced into E. coli for tryptophan-inducible expression (Invitrogen). .. Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Enzyme-linked Immunosorbent Assay:

Article Title: Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice
Article Snippet: Paragraph title: ELISA of proteins in cerebral tissue ... Thereafter, the brain tissue was homogenized in an extraction solution (100 mg of tissue per 1 mL of extraction solution) containing 0.4 mol/L NaCl, 0.05% Tween 20, 0.5% BSA, 0.1 mmol/L phenylmethyl sulfonil fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 20 KI aprotinin, using Ultra-Turrax (Fisher Scientific, Pittsburgh, PA).

Column Chromatography:

Article Title: Click dimers to target HIV TAR RNA conformation
Article Snippet: SC (Sodium Cacodylate), EDTA (Ethylenediamine Tetraacetic Acid), KCl, sodium phosphate (mono- and di-) salts were purchased from Fisher Scientific. .. Silica gel for flash column chromatography was purchased from Sorbent Technologies (Atlanta, GA, USA) as silica gel standard grade (particle size = 40-63 μm).

Produced:

Article Title: Antioxidant Chemistry of Graphene-Based Materials and its Role in Oxidation Protection Technology
Article Snippet: RGO was produced by heating multilayer GO flakes at 250 C for 30 min in nitrogen flow. .. Hydrogen peroxide (H2 O2 ), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA).

Concentration Assay:

Article Title: Barnacle cement: a polymerization model based on evolutionary concepts
Article Snippet: EGTA (Sigma-Aldrich no. E4378) and EDTA (Fisher Chemical, no. BP118) were used. .. The initial concentration of EGTA and EDTA solutions was 1 mg ml–1 (stirred for at least 30 min before use).

Article Title: Temporal changes of cytochrome P450 (Cyp) and eicosanoid-related gene expression in the rat brain after traumatic brain injury
Article Snippet: Probe concentration and quality were assessed by absorbance at 260 nm (A260 ) and the A260 /A280 ratio. .. Sections were washed (50 mM tris, 5 mM EDTA, pH7.4), rinsed in distilled water, dehydrated in increasing ethanol washes, and coverslipped with Permount (Fisher Scientific) for observation under bright field microscopy.

Lysis:

Article Title: β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction
Article Snippet: .. Isolated cardiomyocytes (prepared as described above) were stimulated with 0.1% DMSO, 0.1 µM isoproterenol, or 10 µM ICL1–9 for 5 min. On ice, assay media were removed and 100 μL of lysis buffer was added, cells were scraped and or nutated at 4 °C for 30 min. A total of 20 μL of 6× Laemeli buffer was added and the lysate was boiled for 10 min. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 137 mM NaCl, 10% (vol/vol) glycerol, 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor mixture (Thermo Scientific), and phosphatase inhibitor mixture set IV (Calbiochem). .. Lysates were run on 8% SDS/PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2-mm pore size membranes (Millipore).

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    Fisher Scientific edta
    Time course of reduction of Fab fragment by TCEP agarose The amount of free –SH (cysteine residues) generated by treatment with TCEP agarose after incubation with gentle end-over-end mixing in 100 mM <t>Tris-HCl/5</t> mM <t>EDTA,</t> pH = 8.0 at 22 °C is shown for several samples. Filled squares are from duplicate washed TCEP agarose gel time course experiment samples which were also used to generate the samples analyzed in Fig. 2. Open circle symbols are from samples treated with unwashed TCEP agarose (used as supplied from the manufacturer), while filled triangles are from samples subsequently used for alkylations (using washed TCEP agarose in a variety of buffers after 22 °C incubations from 17 to 24 h) with various reagents. The dotted line represents the 2.0 cysteine/Fab fragment stoichiometry expected if only the targeted disulfide is (quantitatively) reduced. The dashed line represents a power function curve fitted to the two replicate data sets (filled squares) of time course experiments.
    Edta, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific tris acetate ethylenediaminetetraacetic acid edta buffer
    AFM images of DNA origami triangles adsorbed onto boron-implanted silicon substrates ( a – d ) and thermally grown silicon dioxide (SiO 2 ) substrates ( e – h ) as a function of deposition buffer pH. The substrates were cleaned with Piranha + HF. For the pH values below 8.3, the deposition buffer was 10 mM <t>bis-tris</t> HCl with a [MgCl 2 ] of 35 mM. For the pH value of 8.3, 1× tris-acetate- <t>ethylenediaminetetraacetic</t> acid <t>(EDTA)</t> with a [MgCl 2 ] of 35 mM was selected to stay within the buffer range. For all conditions, the deposition incubation time was ~1 h. Scale bars are 500 nm.
    Tris Acetate Ethylenediaminetetraacetic Acid Edta Buffer, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Time course of reduction of Fab fragment by TCEP agarose The amount of free –SH (cysteine residues) generated by treatment with TCEP agarose after incubation with gentle end-over-end mixing in 100 mM Tris-HCl/5 mM EDTA, pH = 8.0 at 22 °C is shown for several samples. Filled squares are from duplicate washed TCEP agarose gel time course experiment samples which were also used to generate the samples analyzed in Fig. 2. Open circle symbols are from samples treated with unwashed TCEP agarose (used as supplied from the manufacturer), while filled triangles are from samples subsequently used for alkylations (using washed TCEP agarose in a variety of buffers after 22 °C incubations from 17 to 24 h) with various reagents. The dotted line represents the 2.0 cysteine/Fab fragment stoichiometry expected if only the targeted disulfide is (quantitatively) reduced. The dashed line represents a power function curve fitted to the two replicate data sets (filled squares) of time course experiments.

    Journal: Biochemical and biophysical research communications

    Article Title: Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

    doi: 10.1016/j.bbrc.2016.10.128

    Figure Lengend Snippet: Time course of reduction of Fab fragment by TCEP agarose The amount of free –SH (cysteine residues) generated by treatment with TCEP agarose after incubation with gentle end-over-end mixing in 100 mM Tris-HCl/5 mM EDTA, pH = 8.0 at 22 °C is shown for several samples. Filled squares are from duplicate washed TCEP agarose gel time course experiment samples which were also used to generate the samples analyzed in Fig. 2. Open circle symbols are from samples treated with unwashed TCEP agarose (used as supplied from the manufacturer), while filled triangles are from samples subsequently used for alkylations (using washed TCEP agarose in a variety of buffers after 22 °C incubations from 17 to 24 h) with various reagents. The dotted line represents the 2.0 cysteine/Fab fragment stoichiometry expected if only the targeted disulfide is (quantitatively) reduced. The dashed line represents a power function curve fitted to the two replicate data sets (filled squares) of time course experiments.

    Article Snippet: Tris base, NaCl, EDTA, and other buffer components, as well as sequencing grade concentrated formic acid, were from Fisher Scientific.

    Techniques: Generated, Incubation, Agarose Gel Electrophoresis

    Fox1 abundance in iron-deficient Chlamydomonas . Protein extracts from C. reinhardtii cells grown in TAP medium containing various concentrations of copper and iron, and collected at a density of 10 7 cells ml −1 , were prepared as described in Materials and Methods. Extracts were analyzed after separation by denaturing gel electrophoresis (7.5% acrylamide), transfer to nitrocellulose (1 h, 100 V, in 25 mM Tris-192 mM glycine-0.04% [wt/vol] SDS-20% [vol/vol] methanol), and incubation with anti-Fox1 antiserum (1:300 dilution). Bound antibody was visualized colorimetrically after incubation with alkaline phosphatase-conjugated secondary antibody. (A) The Fox1 gene product accumulates in −Fe cells. Cells grown in medium containing 200 μM added Fe-EDTA were transferred to fresh medium supplemented with 0.1, 0.25, 1, 18, or 200 μM Fe-EDTA and sampled after 5 days of growth. Protein extracts from 2.5 × 10 6 cells were analyzed as described above. Samples were normalized for loading on the basis of equal cell numbers and verified for accumulation of CF 1 , which is iron independent (see Materials and Methods). Percentages on the left are the fractional amounts of the “0.1 μM Fe” sample that were loaded. (B) Time course of Fox1 induction in −Fe cells. Cells grown with 200 μM added Fe-EDTA were transferred to fresh medium lacking iron (−Fe) or containing 200 μM added Fe-EDTA (+Fe). Cultures were sampled each day for 5 days (d), and ferroxidase abundance was analyzed as described above. Percentages shown on the right arethe amounts of the day 1 sample that were loaded. The accumulation of the α and β subunits of CF 1 is shown as a loading control. (C) Time course of Fox1 mRNA accumulation upon transfer from Fe-replete (200 μM) to Fe-deficent (0 μM) medium. Cultures were sampled at the indicated time after transfer for RNA isolations. The RNAs were analyzed by blot hybridization. The Cβlp mRNA was used as an internal control. Its behavior as a function of cell growth and iron nutrition is typical of many other RNAs. (D) Cu is required for accumulation of Fox1. C. reinhardtii was cultured in copper-supplemented (6 μM, +Cu) or copper-deficient (no added copper, −Cu), iron-supplemented (18 μM, +Fe) or iron-deficient (no added iron, −Fe) TAP medium. One microliter of protein extract from 4 × 10 8 cells was analyzed as described above. Percentages on the left are the fraction of the −Fe-+Cu sample that was applied for quantitation. Molecular masses of markers (Gibco BRL) are indicated in kilodaltons. (E) Cu-dependent accumulation of Fox1 in iron-replete cells. Copper-deficient, iron-replete (18 μM) CC125 cells were transferred to fresh (0 μM supplemental iron) medium containing the indicated amounts of copper and sampled for immunoblot analysis of Fox1 accumulation after 3 days. Each lane was loaded with material from 2 × 10 7 cells (equivalent to approximately 5 μg of chlorophyll). Percentages on the right are the fraction of the sample from medium containing 100 μM supplemental copper.

    Journal: Eukaryotic Cell

    Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii

    doi: 10.1128/EC.1.5.736-757.2002

    Figure Lengend Snippet: Fox1 abundance in iron-deficient Chlamydomonas . Protein extracts from C. reinhardtii cells grown in TAP medium containing various concentrations of copper and iron, and collected at a density of 10 7 cells ml −1 , were prepared as described in Materials and Methods. Extracts were analyzed after separation by denaturing gel electrophoresis (7.5% acrylamide), transfer to nitrocellulose (1 h, 100 V, in 25 mM Tris-192 mM glycine-0.04% [wt/vol] SDS-20% [vol/vol] methanol), and incubation with anti-Fox1 antiserum (1:300 dilution). Bound antibody was visualized colorimetrically after incubation with alkaline phosphatase-conjugated secondary antibody. (A) The Fox1 gene product accumulates in −Fe cells. Cells grown in medium containing 200 μM added Fe-EDTA were transferred to fresh medium supplemented with 0.1, 0.25, 1, 18, or 200 μM Fe-EDTA and sampled after 5 days of growth. Protein extracts from 2.5 × 10 6 cells were analyzed as described above. Samples were normalized for loading on the basis of equal cell numbers and verified for accumulation of CF 1 , which is iron independent (see Materials and Methods). Percentages on the left are the fractional amounts of the “0.1 μM Fe” sample that were loaded. (B) Time course of Fox1 induction in −Fe cells. Cells grown with 200 μM added Fe-EDTA were transferred to fresh medium lacking iron (−Fe) or containing 200 μM added Fe-EDTA (+Fe). Cultures were sampled each day for 5 days (d), and ferroxidase abundance was analyzed as described above. Percentages shown on the right arethe amounts of the day 1 sample that were loaded. The accumulation of the α and β subunits of CF 1 is shown as a loading control. (C) Time course of Fox1 mRNA accumulation upon transfer from Fe-replete (200 μM) to Fe-deficent (0 μM) medium. Cultures were sampled at the indicated time after transfer for RNA isolations. The RNAs were analyzed by blot hybridization. The Cβlp mRNA was used as an internal control. Its behavior as a function of cell growth and iron nutrition is typical of many other RNAs. (D) Cu is required for accumulation of Fox1. C. reinhardtii was cultured in copper-supplemented (6 μM, +Cu) or copper-deficient (no added copper, −Cu), iron-supplemented (18 μM, +Fe) or iron-deficient (no added iron, −Fe) TAP medium. One microliter of protein extract from 4 × 10 8 cells was analyzed as described above. Percentages on the left are the fraction of the −Fe-+Cu sample that was applied for quantitation. Molecular masses of markers (Gibco BRL) are indicated in kilodaltons. (E) Cu-dependent accumulation of Fox1 in iron-replete cells. Copper-deficient, iron-replete (18 μM) CC125 cells were transferred to fresh (0 μM supplemental iron) medium containing the indicated amounts of copper and sampled for immunoblot analysis of Fox1 accumulation after 3 days. Each lane was loaded with material from 2 × 10 7 cells (equivalent to approximately 5 μg of chlorophyll). Percentages on the right are the fraction of the sample from medium containing 100 μM supplemental copper.

    Article Snippet: Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

    Techniques: Nucleic Acid Electrophoresis, Incubation, Hybridization, Cell Culture, Quantitation Assay

    GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Journal: Nanoscale

    Article Title: Antioxidant Chemistry of Graphene-Based Materials and its Role in Oxidation Protection Technology

    doi: 10.1039/c4nr03275f

    Figure Lengend Snippet: GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Article Snippet: Hydrogen peroxide (H2 O2 ), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA).

    Techniques: Generated, Electron Paramagnetic Resonance, Spectroscopy, Concentration Assay, Binding Assay

    AFM images of DNA origami triangles adsorbed onto boron-implanted silicon substrates ( a – d ) and thermally grown silicon dioxide (SiO 2 ) substrates ( e – h ) as a function of deposition buffer pH. The substrates were cleaned with Piranha + HF. For the pH values below 8.3, the deposition buffer was 10 mM bis-tris HCl with a [MgCl 2 ] of 35 mM. For the pH value of 8.3, 1× tris-acetate- ethylenediaminetetraacetic acid (EDTA) with a [MgCl 2 ] of 35 mM was selected to stay within the buffer range. For all conditions, the deposition incubation time was ~1 h. Scale bars are 500 nm.

    Journal: International Journal of Molecular Sciences

    Article Title: Boron-Implanted Silicon Substrates for Physical Adsorption of DNA Origami

    doi: 10.3390/ijms19092513

    Figure Lengend Snippet: AFM images of DNA origami triangles adsorbed onto boron-implanted silicon substrates ( a – d ) and thermally grown silicon dioxide (SiO 2 ) substrates ( e – h ) as a function of deposition buffer pH. The substrates were cleaned with Piranha + HF. For the pH values below 8.3, the deposition buffer was 10 mM bis-tris HCl with a [MgCl 2 ] of 35 mM. For the pH value of 8.3, 1× tris-acetate- ethylenediaminetetraacetic acid (EDTA) with a [MgCl 2 ] of 35 mM was selected to stay within the buffer range. For all conditions, the deposition incubation time was ~1 h. Scale bars are 500 nm.

    Article Snippet: The DNA scaffolds and corresponding staples were mixed in a 1:10 molar ratio, in a 1× tris-acetate-ethylenediaminetetraacetic acid (EDTA) buffer (Fisher Scientific, Hampton, NH, USA), with a pH of 8.3, and a [MgCl2 ] of 12.5 mM.

    Techniques: Incubation