Structured Review

Bio-Rad edta
Mutating the cation-binding residues of SRCR domains abolish function. Through multiple ligand binding assays, we demonstrated the functional importance of calcium binding by the SRCR domains. Mutations affecting site 2 (D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function. (A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a Ca 2+ containing buffer. After extensive washing, bound protein was eluted with <t>EDTA.</t> Eluted fractions were run on a 4 - 20 % <t>SDS-PAGE</t> gel and visualized by Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant forms of SRCR8 were flown over a Heparin (HiTrap HP, 1 ml) column in a Ca 2+ -containing buffer. Protein bound to the column was eluted with 0.5 M EDTA. Only WT SRCR8 bound the Heparin-column. Traces: SRCR8 (blue), D1020A (pink), D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration range of the Spy-2 domain of Spy0843 was coated (0.032 – 3.2 μM). WT and mutant SRCR8 domains were added in molar excess (7.1 μM), and binding was detected with a monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D) Overview of ligand binding studies, + denotes binding, − denotes no binding.
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1) Product Images from "The structure of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR-fold"

Article Title: The structure of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR-fold

Journal: bioRxiv

doi: 10.1101/709915

Mutating the cation-binding residues of SRCR domains abolish function. Through multiple ligand binding assays, we demonstrated the functional importance of calcium binding by the SRCR domains. Mutations affecting site 2 (D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function. (A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a Ca 2+ containing buffer. After extensive washing, bound protein was eluted with EDTA. Eluted fractions were run on a 4 - 20 % SDS-PAGE gel and visualized by Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant forms of SRCR8 were flown over a Heparin (HiTrap HP, 1 ml) column in a Ca 2+ -containing buffer. Protein bound to the column was eluted with 0.5 M EDTA. Only WT SRCR8 bound the Heparin-column. Traces: SRCR8 (blue), D1020A (pink), D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration range of the Spy-2 domain of Spy0843 was coated (0.032 – 3.2 μM). WT and mutant SRCR8 domains were added in molar excess (7.1 μM), and binding was detected with a monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D) Overview of ligand binding studies, + denotes binding, − denotes no binding.
Figure Legend Snippet: Mutating the cation-binding residues of SRCR domains abolish function. Through multiple ligand binding assays, we demonstrated the functional importance of calcium binding by the SRCR domains. Mutations affecting site 2 (D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function. (A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a Ca 2+ containing buffer. After extensive washing, bound protein was eluted with EDTA. Eluted fractions were run on a 4 - 20 % SDS-PAGE gel and visualized by Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant forms of SRCR8 were flown over a Heparin (HiTrap HP, 1 ml) column in a Ca 2+ -containing buffer. Protein bound to the column was eluted with 0.5 M EDTA. Only WT SRCR8 bound the Heparin-column. Traces: SRCR8 (blue), D1020A (pink), D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration range of the Spy-2 domain of Spy0843 was coated (0.032 – 3.2 μM). WT and mutant SRCR8 domains were added in molar excess (7.1 μM), and binding was detected with a monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D) Overview of ligand binding studies, + denotes binding, − denotes no binding.

Techniques Used: Binding Assay, Ligand Binding Assay, Functional Assay, Mutagenesis, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay

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Article Snippet: Cell pellets from single wells of a 24-well plate of infected cells were washed once with PBS and embedded in molten agarose plugs. .. The plugs were incubated in a solution containing SDS, EDTA and proteinase K as described in the protocol provided by Bio-Rad. .. Electrophoresis was performed in 1% agarose gel in CHEF-DR III apparatus (Bio-Rad) at 5.8 V with a switching time gradient of 50–90 s, 120° angle for 22 h at 14 °C.

Filtration:

Article Title: A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations
Article Snippet: Size Exclusion Chromatography Analysis of HsTIM that had been expressed in E. coli and the P1 and P2 proteins were performed in a Superdex 200 10/300GL analytical column (GE Healthcare) on an Ákta FPLC System (GE Healthcare). .. Generally 300 µl of 20 mM triethanolamine, 0.2 mM EDTA, 200 mM NaCl and 1 mM dithiothreitol (pH 7.4) that contained between 25 to 500 µg of protein were applied and eluted with the same buffer. the columns were calibrated with a gel filtration standard (Bio-Rad) containing the following globular protein markers (molecular mass and retention volumes are reported): thyroglobulin (bovine) (669 kDa, 9.8 ml), γ-globulin (bovine) (158 kDa, 12.9 ml), ovalbumin (chicken) (43 kDa, 15.8 ml), myoglobin (horse) (17 kDa, 17.7 ml), and vitamin B12 (1.35 kDa, 21.1 ml). ..

other:

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: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA using Bio-Rad PD10 desalting columns.

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    Bio-Rad tbe buffer
    Optimization and confirmation of human chromatin assembly reaction. a DNA supercoiling assay to optimize the ratio of HsH2A/HsH2B and HsH3.1/HsH4 mRNAs in the chromatin reconstitution reaction for 4 h. b Supercoiling assay of assembled human chromatin. Incubation time indicated with 0, 1, 4, 6, and 8 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones. Supercoils were analyzed by 0.8% agarose <t>TBE</t> gel. Relaxed, linear, and <t>supercoiled</t> DNA are indicated as RC, L, and SC, respectively. c Partial MNase digestion of assembled human chromatin, then samples were run on 2.0% agarose gel. Reconstituted chromatin was digested by MNase for the indicated time (0, 5, 10, 20, 30, and 40 min), and bands corresponding to digestion fragment of the mono-, di-, tri- and tetranucleosome were indicated. Mr: DNA molecular weight marker
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    Bio-Rad edta plasma
    Native <t>PAGE</t> WB analysis of <t>EDTA</t> plasma samples from 31 HAE patients. “ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.
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    Bio-Rad m edta
    Protease protection assays for GR A-NTD, GR C2-NTD, and GR C3-NTD. Trypsin digestions were performed at a protein (1 mg/ml):trypsin mass ratio of 1000:1 at 22 °C in 10 m m <t>HEPES,</t> 80 m m NaCl, 1 m m <t>EDTA,</t> 10% glycerol buffer, pH 7.6 for 0, 5, 10,
    M Edta, supplied by Bio-Rad, 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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    Optimization and confirmation of human chromatin assembly reaction. a DNA supercoiling assay to optimize the ratio of HsH2A/HsH2B and HsH3.1/HsH4 mRNAs in the chromatin reconstitution reaction for 4 h. b Supercoiling assay of assembled human chromatin. Incubation time indicated with 0, 1, 4, 6, and 8 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones. Supercoils were analyzed by 0.8% agarose TBE gel. Relaxed, linear, and supercoiled DNA are indicated as RC, L, and SC, respectively. c Partial MNase digestion of assembled human chromatin, then samples were run on 2.0% agarose gel. Reconstituted chromatin was digested by MNase for the indicated time (0, 5, 10, 20, 30, and 40 min), and bands corresponding to digestion fragment of the mono-, di-, tri- and tetranucleosome were indicated. Mr: DNA molecular weight marker

    Journal: BMC Biotechnology

    Article Title: Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

    doi: 10.1186/s12896-020-00655-6

    Figure Lengend Snippet: Optimization and confirmation of human chromatin assembly reaction. a DNA supercoiling assay to optimize the ratio of HsH2A/HsH2B and HsH3.1/HsH4 mRNAs in the chromatin reconstitution reaction for 4 h. b Supercoiling assay of assembled human chromatin. Incubation time indicated with 0, 1, 4, 6, and 8 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones. Supercoils were analyzed by 0.8% agarose TBE gel. Relaxed, linear, and supercoiled DNA are indicated as RC, L, and SC, respectively. c Partial MNase digestion of assembled human chromatin, then samples were run on 2.0% agarose gel. Reconstituted chromatin was digested by MNase for the indicated time (0, 5, 10, 20, 30, and 40 min), and bands corresponding to digestion fragment of the mono-, di-, tri- and tetranucleosome were indicated. Mr: DNA molecular weight marker

    Article Snippet: Supercoiled plasmid DNAs were separated by 0.8% TBE agarose gel in 0.5x TBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by gel documentation system (Bio-Rad Laboratories).

    Techniques: Incubation, Agarose Gel Electrophoresis, Molecular Weight, Marker

    DNA supercoiling and MNase assays of Drosophila chromatin assembly reactions. a Determination of the appropriate ratio of DmH2A/DmH2B and DmH3/DmH4 mRNAs in the Drosophila chromatin reconstitution reaction. The incubation time was 4 h. Supercoiling assay was performed, samples were analyzed by 0.8% agarose TBE gel. Relaxed, linear, and supercoiled plasmid DNAs are indicated as RC, L, and SC, respectively. b Supercoiling assay of assembled Drosophila chromatin under the indicated reaction time 0, 1, 4, and 6 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones. Supercoiling assay was performed, samples were analyzed by 0.8% agarose TBE gel. c Partial MNase digestion was performed on assembled Drosophila chromatin for the indicated time (0, 5, 10, 20, 30, and 40 min), then samples were run on 2.0% agarose gel. Bands corresponding to digestion fragment of the mono-, di-, and trinucleosomes were detected and indicated. Agarose gels were visualized by ethidium bromide and UV light. Mr: DNA molecular weight marker

    Journal: BMC Biotechnology

    Article Title: Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

    doi: 10.1186/s12896-020-00655-6

    Figure Lengend Snippet: DNA supercoiling and MNase assays of Drosophila chromatin assembly reactions. a Determination of the appropriate ratio of DmH2A/DmH2B and DmH3/DmH4 mRNAs in the Drosophila chromatin reconstitution reaction. The incubation time was 4 h. Supercoiling assay was performed, samples were analyzed by 0.8% agarose TBE gel. Relaxed, linear, and supercoiled plasmid DNAs are indicated as RC, L, and SC, respectively. b Supercoiling assay of assembled Drosophila chromatin under the indicated reaction time 0, 1, 4, and 6 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones. Supercoiling assay was performed, samples were analyzed by 0.8% agarose TBE gel. c Partial MNase digestion was performed on assembled Drosophila chromatin for the indicated time (0, 5, 10, 20, 30, and 40 min), then samples were run on 2.0% agarose gel. Bands corresponding to digestion fragment of the mono-, di-, and trinucleosomes were detected and indicated. Agarose gels were visualized by ethidium bromide and UV light. Mr: DNA molecular weight marker

    Article Snippet: Supercoiled plasmid DNAs were separated by 0.8% TBE agarose gel in 0.5x TBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by gel documentation system (Bio-Rad Laboratories).

    Techniques: Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients. “ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.

    Journal: PLoS ONE

    Article Title: Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

    doi: 10.1371/journal.pone.0112051

    Figure Lengend Snippet: Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients. “ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.

    Article Snippet: Native PAGE WB analysis of patient samples and controls Patient EDTA plasma samples were thawed at 24°C for 25 minutes, and diluted to 25% (v/v) in PBS buffer, and further diluted 50% (v/v) in native sample buffer (Bio-Rad), giving a final plasma dilution of 10% (v/v). pC1-inh (1 µg/lane), LMW pC1-inh (60 ng/lane), C1-inh (24 ng/lane) and the EDTA plasma pool (10% (v/v)) were used for controls.

    Techniques: Clear Native PAGE, Western Blot, Mutagenesis, Molecular Weight, Filtration, Ion Exchange Chromatography

    Native PAGE WB analysis of EDTA plasma samples from 34 healthy individuals, and one polymer positive HAE patient (lab. nr. 33: CPDA plasma). “Norm. nr.”. depicts the number ascribed to each healthy individual, “P”, “M” and “ID. no.” depictions as for figure 3 .

    Journal: PLoS ONE

    Article Title: Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

    doi: 10.1371/journal.pone.0112051

    Figure Lengend Snippet: Native PAGE WB analysis of EDTA plasma samples from 34 healthy individuals, and one polymer positive HAE patient (lab. nr. 33: CPDA plasma). “Norm. nr.”. depicts the number ascribed to each healthy individual, “P”, “M” and “ID. no.” depictions as for figure 3 .

    Article Snippet: Native PAGE WB analysis of patient samples and controls Patient EDTA plasma samples were thawed at 24°C for 25 minutes, and diluted to 25% (v/v) in PBS buffer, and further diluted 50% (v/v) in native sample buffer (Bio-Rad), giving a final plasma dilution of 10% (v/v). pC1-inh (1 µg/lane), LMW pC1-inh (60 ng/lane), C1-inh (24 ng/lane) and the EDTA plasma pool (10% (v/v)) were used for controls.

    Techniques: Clear Native PAGE, Western Blot

    Native PAGE analysis of EDTA plasma samples from six HAE patients, representing three different genotypes. Nomenclature used as for figure 3 .

    Journal: PLoS ONE

    Article Title: Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

    doi: 10.1371/journal.pone.0112051

    Figure Lengend Snippet: Native PAGE analysis of EDTA plasma samples from six HAE patients, representing three different genotypes. Nomenclature used as for figure 3 .

    Article Snippet: Native PAGE WB analysis of patient samples and controls Patient EDTA plasma samples were thawed at 24°C for 25 minutes, and diluted to 25% (v/v) in PBS buffer, and further diluted 50% (v/v) in native sample buffer (Bio-Rad), giving a final plasma dilution of 10% (v/v). pC1-inh (1 µg/lane), LMW pC1-inh (60 ng/lane), C1-inh (24 ng/lane) and the EDTA plasma pool (10% (v/v)) were used for controls.

    Techniques: Clear Native PAGE

    Protease protection assays for GR A-NTD, GR C2-NTD, and GR C3-NTD. Trypsin digestions were performed at a protein (1 mg/ml):trypsin mass ratio of 1000:1 at 22 °C in 10 m m HEPES, 80 m m NaCl, 1 m m EDTA, 10% glycerol buffer, pH 7.6 for 0, 5, 10,

    Journal: The Journal of Biological Chemistry

    Article Title: Thermodynamic Dissection of the Intrinsically Disordered N-terminal Domain of Human Glucocorticoid Receptor *

    doi: 10.1074/jbc.M112.355651

    Figure Lengend Snippet: Protease protection assays for GR A-NTD, GR C2-NTD, and GR C3-NTD. Trypsin digestions were performed at a protein (1 mg/ml):trypsin mass ratio of 1000:1 at 22 °C in 10 m m HEPES, 80 m m NaCl, 1 m m EDTA, 10% glycerol buffer, pH 7.6 for 0, 5, 10,

    Article Snippet: Digestions were performed at a protein:trypsin mass ratio of 1000:1 in 10 m m HEPES, 80 m m NaCl, 1 m m EDTA, 10% glycerol buffer, pH 7.6 for 0, 5, 10, and 30 min. Digestions were quenched by mixing the protein and trypsin mixture with 6× Laemmli sample buffer and boiling at 100 °C for 10 min. A 40-μg sample at each time point was separated on 4–15% Tris-HCl gel (Bio-Rad) with SDS-Tris-glycine gel running buffer.

    Techniques:

    Reaction of the reduced WT ThyX·5-dUMPS complex with CH 2 THF. Anaerobic solutions of reduced WT ThyX (14 μM active sites) and dUMP [300 μM (black)] or with 5-dUMPS [300 μM (blue)] were mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C.

    Journal: Biochemistry

    Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation

    doi: 10.1021/bi500648n

    Figure Lengend Snippet: Reaction of the reduced WT ThyX·5-dUMPS complex with CH 2 THF. Anaerobic solutions of reduced WT ThyX (14 μM active sites) and dUMP [300 μM (black)] or with 5-dUMPS [300 μM (blue)] were mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C.

    Article Snippet: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA using Bio-Rad PD10 desalting columns.

    Techniques: Flow Cytometry, Spectrophotometry

    Spectrum of the intermediate detected in the oxidative half-reaction. An anaerobic solution of reduced WT ThyX (14 μM active sites, after mixing) and dUMP (300 μM) in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA was mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer in diode-array mode. (A) Stereoview of spectra as a function of time. Spectra were recorded with an integration time of 1.5 ms at various intervals out to 10 s. Note the logarithmic time scale. (B) Deconvoluted intermediate spectra calculated by singular-value decomposition using the raw data in panel A and a two-step mechanism, giving rate constants of 28.4 and 0.2 s –1 .

    Journal: Biochemistry

    Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation

    doi: 10.1021/bi500648n

    Figure Lengend Snippet: Spectrum of the intermediate detected in the oxidative half-reaction. An anaerobic solution of reduced WT ThyX (14 μM active sites, after mixing) and dUMP (300 μM) in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA was mixed with 400 μM CH 2 THF and 15 mM formaldehyde using a stopped-flow spectrophotometer in diode-array mode. (A) Stereoview of spectra as a function of time. Spectra were recorded with an integration time of 1.5 ms at various intervals out to 10 s. Note the logarithmic time scale. (B) Deconvoluted intermediate spectra calculated by singular-value decomposition using the raw data in panel A and a two-step mechanism, giving rate constants of 28.4 and 0.2 s –1 .

    Article Snippet: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA using Bio-Rad PD10 desalting columns.

    Techniques: Flow Cytometry, Spectrophotometry, Mass Spectrometry

    Chemical quenching. (A) An anaerobic solution of reduced WT ThyX (50 μM) and dUMP (300 μM) was mixed with 400 μM CH 2 THF and 15 mM formaldehyde in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA. The reaction was quenched with 1 M HCl at different times, and the concentrations of dUMP (green) and dTMP (orange) were calculated from the area under the peaks of HPLC chromatograms. An absorbance trace at 420 nm obtained in stopped-flow experiments (black) is shown for comparison. The vertical lines at 0.13 and 2.6 s indicate the times of maximal accumulation of intermediates I 1 , detected by the flavin spectral change, and I 2 , detected by the consumption of dUMP. (B) Calculated concentrations of species during the oxidative half-reaction. The rate constants from stopped-flow experiments, 28.4 and 0.2 s –1 , and the rate constant from the consumption of dUMP, 0.7 s –1 , observed by quenching, were used to simulate consecutive reactions. The simulation used an enzyme concentration of 20 μM and shows that intermediates accumulate maximally at 0.13 and 2.6 s.

    Journal: Biochemistry

    Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation

    doi: 10.1021/bi500648n

    Figure Lengend Snippet: Chemical quenching. (A) An anaerobic solution of reduced WT ThyX (50 μM) and dUMP (300 μM) was mixed with 400 μM CH 2 THF and 15 mM formaldehyde in 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA. The reaction was quenched with 1 M HCl at different times, and the concentrations of dUMP (green) and dTMP (orange) were calculated from the area under the peaks of HPLC chromatograms. An absorbance trace at 420 nm obtained in stopped-flow experiments (black) is shown for comparison. The vertical lines at 0.13 and 2.6 s indicate the times of maximal accumulation of intermediates I 1 , detected by the flavin spectral change, and I 2 , detected by the consumption of dUMP. (B) Calculated concentrations of species during the oxidative half-reaction. The rate constants from stopped-flow experiments, 28.4 and 0.2 s –1 , and the rate constant from the consumption of dUMP, 0.7 s –1 , observed by quenching, were used to simulate consecutive reactions. The simulation used an enzyme concentration of 20 μM and shows that intermediates accumulate maximally at 0.13 and 2.6 s.

    Article Snippet: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA using Bio-Rad PD10 desalting columns.

    Techniques: High Performance Liquid Chromatography, Flow Cytometry, Concentration Assay

    Oxidative half-reactions of variant enzymes. The reduced variant enzyme·dUMP complexes were mixed with saturating concentrations CH 2 THF using a stopped-flow spectrophotometer. The reactions were monitored by their absorbance at 420 nm. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C. Traces, labeled by ThyX variant, are displayed in two panels for the sake of clarity.

    Journal: Biochemistry

    Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation

    doi: 10.1021/bi500648n

    Figure Lengend Snippet: Oxidative half-reactions of variant enzymes. The reduced variant enzyme·dUMP complexes were mixed with saturating concentrations CH 2 THF using a stopped-flow spectrophotometer. The reactions were monitored by their absorbance at 420 nm. The reaction mixtures were in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH 2 O at 25 °C. Traces, labeled by ThyX variant, are displayed in two panels for the sake of clarity.

    Article Snippet: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA using Bio-Rad PD10 desalting columns.

    Techniques: Variant Assay, Flow Cytometry, Spectrophotometry, Labeling