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  • 99
    Thermo Fisher tris
    HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), <t>MOPS-NaOH</t> buffer (circles), <t>Tris–HCl</t> buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.
    Tris, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris
    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml <t>clathrin-Sepharose</t> in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M <t>Tris</t> (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).
    Tris, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 38479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 38479 article reviews
    Price from $9.99 to $1999.99
    tris - by Bioz Stars, 2020-08
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    99
    Bio-Rad tris
    Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% <t>Bis-Tris</t> protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM <t>NaCl,</t> 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p
    Tris, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 4105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher agent tris
    Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% <t>Bis-Tris</t> protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM <t>NaCl,</t> 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p
    Agent Tris, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tris base
    (Panel A ) Minocycline (20 µM) in the presence of 3 mM Ca 2+ induces an electrical current in BLM made from DPhPC. The experimental buffer was 10 mM <t>Tris,</t> 10 mM <t>MES,</t> 100 mM KCl, pH 8.5; the voltage was 30 mV. (Panels B, C ) Ion channel activity induced by minocycline (8 µM) in the presence of Ca 2+ (3 mM in panel B and 20 µM in panel C) at 50 mV in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 1 M KCl, pH 9.0. (A current of 0.7 pA at 50 mV corresponds to a conductance of 14 pS)
    Tris Base, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore tris acetate
    ( A ) ChIP experiment to determine the occupancy of hras -1, hras -2 and control sequence (870 bp downstream from TSS) by <t>hnRNP</t> A1. Histograms shows the relative occupancy of hras -1 and hras -2 by hnRNP A1, RNA Pol II (positive control) and IgG (negative control). Data have been normalized by IgG signal; ( B ) EMSA of 32 P-labelled hras -1 Y and hras -2 Y in 50 mM <t>Tris-acetate</t> pH 5.5, 50 mM KCl, incubated 40 min at room temperature with increasing amounts of recombinant hnRNP A1 (0–12 μg). Lane (Δ,A1) indicates the i M incubated 40 min at room temperature, with denatured hnRNPA1 in binding buffer (see Methods); ( C ) EMSA at pH 5.5 of hras -1 Y with BSA or denatured hnRNP A1 and EMSA of hras -1 Y (m) with hnRNP A1; ss = single-stranded oligonucleotide; 1:1 and 1:2 DNA-protein complexes.
    Tris Acetate, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), MOPS-NaOH buffer (circles), Tris–HCl buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.

    Journal: Nucleic Acids Research

    Article Title: Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

    doi: 10.1093/nar/gkh743

    Figure Lengend Snippet: HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), MOPS-NaOH buffer (circles), Tris–HCl buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.

    Article Snippet: 3-(Cyclohexylamino)propane sulfonic acid (CAPS), N -Tris(hydroxymethyl)methylglycine (TRICINE), Tris, MOPS, PIPES and morpholinoethane sulfonic acid (MES) buffers were each prepared at the same ionic strength and treated with RNAsecure reagent (Ambion) prior to use.

    Techniques: Incubation

    Effect of pH on HCV helicase catalyzed ATP hydrolysis. ( A ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the absence of nucleic acid as a function of pH. ( B ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the presence of saturating 2 mM poly(U) RNA. In both (A) and (B), reactions were performed in MES buffer (squares), PIPES buffer (triangles), MOPS buffer (diamonds), TRIS buffer (circles), Tricene buffer (×) or CAPS buffer (+). Average rate constants from four separate determinations are shown with the standard deviations as error bars.

    Journal: Nucleic Acids Research

    Article Title: Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

    doi: 10.1093/nar/gkh743

    Figure Lengend Snippet: Effect of pH on HCV helicase catalyzed ATP hydrolysis. ( A ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the absence of nucleic acid as a function of pH. ( B ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the presence of saturating 2 mM poly(U) RNA. In both (A) and (B), reactions were performed in MES buffer (squares), PIPES buffer (triangles), MOPS buffer (diamonds), TRIS buffer (circles), Tricene buffer (×) or CAPS buffer (+). Average rate constants from four separate determinations are shown with the standard deviations as error bars.

    Article Snippet: 3-(Cyclohexylamino)propane sulfonic acid (CAPS), N -Tris(hydroxymethyl)methylglycine (TRICINE), Tris, MOPS, PIPES and morpholinoethane sulfonic acid (MES) buffers were each prepared at the same ionic strength and treated with RNAsecure reagent (Ambion) prior to use.

    Techniques:

    Fluorescence spectra of 10 μM α-syn only (solid) and mixture of 10 μM α-syn and 500 μM Fe(II) before (dashed) and after (dotted) aeration in Tris buffer at pH 7.4.

    Journal: Journal of inorganic biochemistry

    Article Title: Binding of ?-Synuclein with Fe(III) and with Fe(II) and Biological Implications of the Resultant Complexes

    doi: 10.1016/j.jinorgbio.2009.11.005

    Figure Lengend Snippet: Fluorescence spectra of 10 μM α-syn only (solid) and mixture of 10 μM α-syn and 500 μM Fe(II) before (dashed) and after (dotted) aeration in Tris buffer at pH 7.4.

    Article Snippet: Sodium dihydrogen phosphate, sodium hydroxide, sodium sulfate, ammonium sulfate, trifluoroacetic acid, ferrous ammonium sulfate (Fe(NH4 )2 (SO4 )2 ), Trizma base (Tris), isopropyl β-D-thiogalactopyranoside (IPTG), and acetonitrile were purchased from Thermo Fisher Scientific Inc. (Pittsburgh, PA).

    Techniques: Fluorescence

    (A) Cyclic voltammograms of Tris buffer (pH 7.4) containing 200 μM α-syn (dotted line curve) in ambient atmosphere, 200 μM α-syn and 200 μM Fe(II) (solid line curve) in a N 2 -purged glovebox, and 200 μM α-syn and 200 μM Fe(II) after exposure to air for 2 h (dashed line curve). The scan rate was 20 mV/s and the solid arrow indicates the initial scan direction. The dashed arrow indicates a shoulder peak at −0.075 V. Inset: a CV of Fe(NH 4 ) 2 (SO 4 ) 2 . (B) Differential pulse votammograms of 200 μM α-syn in the presence of various concentration of Fe(NH 4 ) 2 (SO 4 ) 2 : 200 μM (solid line curve), 400 μM (dotted line curve), 800 μM (dashed line curve), and 1000 μM (dash-dotted line curve).

    Journal: Journal of inorganic biochemistry

    Article Title: Binding of ?-Synuclein with Fe(III) and with Fe(II) and Biological Implications of the Resultant Complexes

    doi: 10.1016/j.jinorgbio.2009.11.005

    Figure Lengend Snippet: (A) Cyclic voltammograms of Tris buffer (pH 7.4) containing 200 μM α-syn (dotted line curve) in ambient atmosphere, 200 μM α-syn and 200 μM Fe(II) (solid line curve) in a N 2 -purged glovebox, and 200 μM α-syn and 200 μM Fe(II) after exposure to air for 2 h (dashed line curve). The scan rate was 20 mV/s and the solid arrow indicates the initial scan direction. The dashed arrow indicates a shoulder peak at −0.075 V. Inset: a CV of Fe(NH 4 ) 2 (SO 4 ) 2 . (B) Differential pulse votammograms of 200 μM α-syn in the presence of various concentration of Fe(NH 4 ) 2 (SO 4 ) 2 : 200 μM (solid line curve), 400 μM (dotted line curve), 800 μM (dashed line curve), and 1000 μM (dash-dotted line curve).

    Article Snippet: Sodium dihydrogen phosphate, sodium hydroxide, sodium sulfate, ammonium sulfate, trifluoroacetic acid, ferrous ammonium sulfate (Fe(NH4 )2 (SO4 )2 ), Trizma base (Tris), isopropyl β-D-thiogalactopyranoside (IPTG), and acetonitrile were purchased from Thermo Fisher Scientific Inc. (Pittsburgh, PA).

    Techniques: Concentration Assay

    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Journal: Journal of neuroscience research

    Article Title: Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission

    doi: 10.1002/jnr.490410104

    Figure Lengend Snippet: The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Article Snippet: The Sepharose was then eluted three times with 0.5 ml 0.5 M Tris, 0.1 mM PMSF (pH 7.0) at 37°C for 15 min. Flow-through, washes, and eluates were all concentrated using Millipore quick concentrator-10s, and each sample was brought to 20 µl in IX SDS sample buffer.

    Techniques: Incubation, Isolation, Binding Assay, Flow Cytometry, SDS Page, Silver Staining

    Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p

    Journal: PLoS Pathogens

    Article Title: Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection

    doi: 10.1371/journal.ppat.1007006

    Figure Lengend Snippet: Biochemical characterization of the recombinant protein LuloHya. (A) Purification of LuloHya by size exclusion chromatography using Superdex 200 Increase 10/300 GL column. (B) Coomassie-stained gel electrophoresis of LuloHya (1 μg). Mouse anti-LuloHya antibodies (1:5,000) recognized LuloHya (100 ng) and a single band in the SGE (5 pairs of SG) by Western blot. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) NuPAGE Novex 4–12% Bis-Tris protein gel shows differences in electrophoretic pattern of LuloHya (1 μg) and its deglycosylated form (deLuloHya: 1 μg) which runs at the expected molecular weight (42.3 kDa). M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (D) Hyaluronidase activity of 10 nM LuloHya and its deglycosylated form (deLuloHya). As negative controls, 4 micrograms of HA were incubated with TBS instead of recombinant protein or the deglycosylation enzyme mix (DeglycoMx Kit, QABio) without protein. (E) Turbidimetric assay showed a clear pH dependence of hyaluronidase activity of LuloHya. Reaction mixtures were prepared with solutions containing 25 mM buffer (described in Methods), 100 mM NaCl, 0.1% BSA, and different pH values (4–12.5; adjusted with a pHmeter 430, Corning). (F) Ionic strength dependency was analyzed in reaction mixtures of 25 mM HEPES, 0.1% BSA, pH 7.3 with variable NaCl concentration (25–1000 mM). Hyaluronidase activity is inversely expressed as the remaining HA (%) after treatment of 4 μg of HA with 10 nM enzyme during 1 h at 37°C. Biological triplicates and technical duplicates were assessed. Multiple comparisons were done by one-way ANOVA (****: p

    Article Snippet: Proteins were transferred to a nitrocellulose membrane (iBlot, Invitrogene) that was blocked overnight at 4°C with blocking buffer: 50 mM Tris, 150 mM NaCl containing 5% (w/v) powdered nonfat blotting-grade milk (Bio-Rad) and 0.05% of Tween-20 (Sigma).

    Techniques: Recombinant, Purification, Size-exclusion Chromatography, Staining, Nucleic Acid Electrophoresis, Western Blot, Molecular Weight, Activity Assay, Incubation, Turbidimetric Assay, Concentration Assay

    (Panel A ) Minocycline (20 µM) in the presence of 3 mM Ca 2+ induces an electrical current in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 100 mM KCl, pH 8.5; the voltage was 30 mV. (Panels B, C ) Ion channel activity induced by minocycline (8 µM) in the presence of Ca 2+ (3 mM in panel B and 20 µM in panel C) at 50 mV in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 1 M KCl, pH 9.0. (A current of 0.7 pA at 50 mV corresponds to a conductance of 14 pS)

    Journal: Journal of bioenergetics and biomembranes

    Article Title: Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels

    doi: 10.1007/s10863-010-9271-1

    Figure Lengend Snippet: (Panel A ) Minocycline (20 µM) in the presence of 3 mM Ca 2+ induces an electrical current in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 100 mM KCl, pH 8.5; the voltage was 30 mV. (Panels B, C ) Ion channel activity induced by minocycline (8 µM) in the presence of Ca 2+ (3 mM in panel B and 20 µM in panel C) at 50 mV in BLM made from DPhPC. The experimental buffer was 10 mM Tris, 10 mM MES, 1 M KCl, pH 9.0. (A current of 0.7 pA at 50 mV corresponds to a conductance of 14 pS)

    Article Snippet: β-Alanine, micro Biuret protein assay kit, bovine serum albumin (fatty acid free grade Sigma A-6003), CaCl2 , EGTA, gramicidin A, Hepes (ultra grade), minocycline, KCl, KOH, KH2 PO4 , MES, Ruthenium 360 (Ru360), succinate, sucrose (ultra grade), PBS-Tween buffer, and Tris-base were obtained from Sigma (St. Louis, MO, USA).

    Techniques: Activity Assay

    ( A ) ChIP experiment to determine the occupancy of hras -1, hras -2 and control sequence (870 bp downstream from TSS) by hnRNP A1. Histograms shows the relative occupancy of hras -1 and hras -2 by hnRNP A1, RNA Pol II (positive control) and IgG (negative control). Data have been normalized by IgG signal; ( B ) EMSA of 32 P-labelled hras -1 Y and hras -2 Y in 50 mM Tris-acetate pH 5.5, 50 mM KCl, incubated 40 min at room temperature with increasing amounts of recombinant hnRNP A1 (0–12 μg). Lane (Δ,A1) indicates the i M incubated 40 min at room temperature, with denatured hnRNPA1 in binding buffer (see Methods); ( C ) EMSA at pH 5.5 of hras -1 Y with BSA or denatured hnRNP A1 and EMSA of hras -1 Y (m) with hnRNP A1; ss = single-stranded oligonucleotide; 1:1 and 1:2 DNA-protein complexes.

    Journal: Scientific Reports

    Article Title: GC-elements controlling HRAS transcription form i-motif structures unfolded by heterogeneous ribonucleoprotein particle A1

    doi: 10.1038/srep18097

    Figure Lengend Snippet: ( A ) ChIP experiment to determine the occupancy of hras -1, hras -2 and control sequence (870 bp downstream from TSS) by hnRNP A1. Histograms shows the relative occupancy of hras -1 and hras -2 by hnRNP A1, RNA Pol II (positive control) and IgG (negative control). Data have been normalized by IgG signal; ( B ) EMSA of 32 P-labelled hras -1 Y and hras -2 Y in 50 mM Tris-acetate pH 5.5, 50 mM KCl, incubated 40 min at room temperature with increasing amounts of recombinant hnRNP A1 (0–12 μg). Lane (Δ,A1) indicates the i M incubated 40 min at room temperature, with denatured hnRNPA1 in binding buffer (see Methods); ( C ) EMSA at pH 5.5 of hras -1 Y with BSA or denatured hnRNP A1 and EMSA of hras -1 Y (m) with hnRNP A1; ss = single-stranded oligonucleotide; 1:1 and 1:2 DNA-protein complexes.

    Article Snippet: Radiolabelled oligonucleotides (10 nM) were incubated for 30 min at 20 °C with increasing amounts of hnRNP A1 (0–12 μg) as specified in , in 50 mM Tris–acetate, pH 5.5, 50 mM KCl, 1 mM DTT, 8% glycerol, 1% Phosphatase Inhibitor Cocktail I (Sigma, Milan, Italy), 5 mM NaF, 1 mM Na3 VO4 , 2.5 ng/μl salmon sperm DNA (binding buffer).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Positive Control, Negative Control, Incubation, Recombinant, Binding Assay

    Circular dichroism analysis of 3 μM (0.5 cm pathlength cell) hras -1 Y and hras -2 Y at pH 5.5, 50 mM Tris-acetate, 50 mM KCl, after incubation with increasing amounts of hnRNP A1 (r = 0–4). Spectra of DNA-protein complex have been subtracted of protein spectrum.

    Journal: Scientific Reports

    Article Title: GC-elements controlling HRAS transcription form i-motif structures unfolded by heterogeneous ribonucleoprotein particle A1

    doi: 10.1038/srep18097

    Figure Lengend Snippet: Circular dichroism analysis of 3 μM (0.5 cm pathlength cell) hras -1 Y and hras -2 Y at pH 5.5, 50 mM Tris-acetate, 50 mM KCl, after incubation with increasing amounts of hnRNP A1 (r = 0–4). Spectra of DNA-protein complex have been subtracted of protein spectrum.

    Article Snippet: Radiolabelled oligonucleotides (10 nM) were incubated for 30 min at 20 °C with increasing amounts of hnRNP A1 (0–12 μg) as specified in , in 50 mM Tris–acetate, pH 5.5, 50 mM KCl, 1 mM DTT, 8% glycerol, 1% Phosphatase Inhibitor Cocktail I (Sigma, Milan, Italy), 5 mM NaF, 1 mM Na3 VO4 , 2.5 ng/μl salmon sperm DNA (binding buffer).

    Techniques: Incubation

    ( A ) Sequences of the GC-rich elements located in the HRAS promoter upstream of major TSS’s; ( B,C ) Circular dichroism titrations of hras -1 Y and hras -2 Y (3 μM, 1 cm pathlength cell) in 50 mM Tris-acetate, 50 mM KCl, 40% PEG-300 and pH from 4.5 to 8; ( D ) Ellipticity (287 nm) versus pH curves for hras -1 Y and hras -2 Y in the presence and absence of PEG-300; ( E ) Determination of number of protons picked up by hras -1 Y and hras -2 Y upon folding into the i M.

    Journal: Scientific Reports

    Article Title: GC-elements controlling HRAS transcription form i-motif structures unfolded by heterogeneous ribonucleoprotein particle A1

    doi: 10.1038/srep18097

    Figure Lengend Snippet: ( A ) Sequences of the GC-rich elements located in the HRAS promoter upstream of major TSS’s; ( B,C ) Circular dichroism titrations of hras -1 Y and hras -2 Y (3 μM, 1 cm pathlength cell) in 50 mM Tris-acetate, 50 mM KCl, 40% PEG-300 and pH from 4.5 to 8; ( D ) Ellipticity (287 nm) versus pH curves for hras -1 Y and hras -2 Y in the presence and absence of PEG-300; ( E ) Determination of number of protons picked up by hras -1 Y and hras -2 Y upon folding into the i M.

    Article Snippet: Radiolabelled oligonucleotides (10 nM) were incubated for 30 min at 20 °C with increasing amounts of hnRNP A1 (0–12 μg) as specified in , in 50 mM Tris–acetate, pH 5.5, 50 mM KCl, 1 mM DTT, 8% glycerol, 1% Phosphatase Inhibitor Cocktail I (Sigma, Milan, Italy), 5 mM NaF, 1 mM Na3 VO4 , 2.5 ng/μl salmon sperm DNA (binding buffer).

    Techniques: