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    Structured Review

    Thermo Fisher m dithiothreitol
    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) <t>dithiothreitol.</t> Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.
    M Dithiothreitol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Redox Regulation of Methionine Aminopeptidase 2 Activity *"

    Article Title: Redox Regulation of Methionine Aminopeptidase 2 Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.554253

    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.
    Figure Legend Snippet: Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.

    Techniques Used: Labeling, SDS Page

    Related Articles

    Centrifugation:

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    Article Snippet: Next, 0.3 mg of ScFvEGFR was washed by centrifugation using a 10 K Nanosep® centrifuge tube (Pall Corporation, Putnam, CT, USA). .. Sulfo-LC-SPDP-modified antibody was treated with dithiothreitol (50 mM), a reducing reagent, for 30 minutes followed by use of a desalting column (Zeba desalt spin columns, Pierce Biotechnology) to remove the excess dithiothreitol.

    Luciferase:

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    Article Snippet: The activity of firefly luciferase was measured by diluting samples 250-fold in 20 mM HEPES pH 7.2, 50 mM potassium chloride, 5 mM magnesium chloride, 2 mM dithiothreitol, 350 μM ATP. .. 100 μl of these diluted samples was then mixed with 35 μl of a luciferin mixture consisting of 20 mM HEPES pH 7.2, 5 mM magnesium chloride, 2 mM dithiothreitol, 0.4 mg/mL bovine serum albumin, and 0.5 mM luciferin (Invitrogen).

    Filtration:

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    Mass Spectrometry:

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    Article Snippet: The purity of the peptides was > 98%, and their molecular masses were assessed by MALDI-TOF MS on a Shimadzu AXIMA-TOF . .. The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    TRAP Assay:

    Article Title: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis
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    Synthesized:

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase
    Article Snippet: Peptide synthesis The artificial WSTF PHD_EL5 RING finger and its five mutants in which Lys residues were replaced with Arg (K4R, K8R, K9R, K14R, and K23R) were synthesized by the standard F-moc solid-phase method ( ). .. The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    Construct:

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    Electrophoresis:

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    Article Snippet: Dithiothreitol was added to a final concentration of 50 mM with the remaining sample volume made up with 1× Bolt sample buffer from Life Technologies. .. Proteins were separated by SDS-PAGE electrophoresis in 4–12 % Bis Tris Plus 17 well polyacrylamide gels from Life Technologies.

    Article Title: Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells
    Article Snippet: SDS-PAGE for tryptic digestion Samples were boiled in 2% SDS-loading buffer with dithiothreitol and subjected to SDS-PAGE using a NuPAGE Novex Bis-Tris Mini gel (4–12%) with NuPAGE MOPS SDS-Running buffer supplemented with 1× NuPAGE antioxidant (Life Technologies). .. After electrophoresis, the gel was stained with InstantBlue protein stain and cut into approximately 2 × 6 mm slices then each slice further cut into 1 mm2 pieces.

    Incubation:

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    Article Snippet: Samples were then washed in 1% FSG in PBS, 0.05% TX-100, and incubated with 50 µl of 1:500 dilution of Alexa-fluor 488-conjugated goat anti-mouse IgG and Alexa-fluor 594-conjugated goat anti-rabbit IgG for 1 h. Coverslips were washed and mounted on glass slides, sealed with Hydromount containing 2.5% DABCO and left to set in the dark and sealed with nail varnish. .. Bloodstream-form cells (5 × 106 ) were lysed in SDS-sample buffer, 0.1 M dithiothreitol and run on a 4–12% Nupage gel with MOPS running buffer (Invitrogen), transferred to nitrocellulose and developed using 1:1000 mouse anti-TbPMI, 1:100 000 anti-mouse horseradish peroxidase conjugate (Stratech, UK) and West Pico ECL (Pierce/ThermoScientific) and Hyperfilm ECL film (GE Healthcare, UK).

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    Article Snippet: The IONP suspension was then added with 25 μL of fresh prepared 20 mM Sulfo-LC-SPDP solution and incubated for 40 minutes at room temperature. .. Sulfo-LC-SPDP-modified antibody was treated with dithiothreitol (50 mM), a reducing reagent, for 30 minutes followed by use of a desalting column (Zeba desalt spin columns, Pierce Biotechnology) to remove the excess dithiothreitol.

    Article Title: Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an E. coli cell-free system
    Article Snippet: 100 μl of these diluted samples was then mixed with 35 μl of a luciferin mixture consisting of 20 mM HEPES pH 7.2, 5 mM magnesium chloride, 2 mM dithiothreitol, 0.4 mg/mL bovine serum albumin, and 0.5 mM luciferin (Invitrogen). .. 10 μl of these dilutions was then mixed with 10 μl of 8 mg/mL ONPG and incubated at 37 °C for 15 minutes.

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    Article Snippet: .. Subsequently, the RT master mix containing 4 μl of 5x first strand buffer, 1 μl 0.1 M dithiothreitol, 1 μl dNTP mix (10 mM each), 1 μl Superscript IV reverse transcriptase (200 U/μl, Invitrogen), 1 μl RNasin Plus RNase inhibitor (40 U/μl, Promega) was added and reactions were incubated at 60°C for 10 minutes followed by heating at 80°C for 10 minutes. ..

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    Article Snippet: .. A master mixture comprised of 6 μl of 5× Superscript II buffer, 2 μl of deoxynucleoside triphosphate mixture (2.5 mM each), 3 μl of 0.1 M dithiothreitol, and 1 μl of Superscript II reverse transcriptase (Invitrogen; 200 U/μl) was added to the RNA solution (final volume, 30 μl), and the mixture was incubated at 42°C for 2 h. A control sample that contained RNA and all of the RT components except the Superscript II reverse transcriptase was prepared simultaneously. .. The PCRs were then performed using undiluted RNA control sample, serial dilution of cDNA (1, 1:10 and 1:100), and a gDNA template control.

    Activity Assay:

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    Expressing:

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    BIA-KA:

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    Modification:

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    Western Blot:

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    Article Title: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis
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    High Performance Liquid Chromatography:

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    Article Snippet: After cleavage with trifluoroacetic acid, peptide purification was performed on a reverse-phase HPLC with Shim-pack C18 column equipment (Shimadzu Corp.). .. The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    Conjugation Assay:

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    Transfection:

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    Article Snippet: After 48 h of transfection, total cell extracts were prepared in 1% Triton buffer in PBS, according to the Western blot protocol described above. .. The samples were treated with or without reducing agent 100 mM dithiothreitol (DTT) (Thermo Fisher Scientific, R0861). (ii) SOD1 aggregate formation was verified by filter trap assay as detailed in [ ].

    Sequencing:

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    Serial Dilution:

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    Article Snippet: A master mixture comprised of 6 μl of 5× Superscript II buffer, 2 μl of deoxynucleoside triphosphate mixture (2.5 mM each), 3 μl of 0.1 M dithiothreitol, and 1 μl of Superscript II reverse transcriptase (Invitrogen; 200 U/μl) was added to the RNA solution (final volume, 30 μl), and the mixture was incubated at 42°C for 2 h. A control sample that contained RNA and all of the RT components except the Superscript II reverse transcriptase was prepared simultaneously. .. The PCRs were then performed using undiluted RNA control sample, serial dilution of cDNA (1, 1:10 and 1:100), and a gDNA template control.

    Generated:

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    Article Snippet: RNA extraction & reverse transcription Total RNA was extracted from DL221, MLS1765-92 cell lines and experimentally generated cancer-associated fibroblasts using the RNeasy Mini Kit, including DNase treatment (Qiagen). .. 100 U SuperScript III, 20 U RNaseOUT, 5 mM dithiothreitol, 50 mM Tris-HCl, pH 8.3, 75 mM KCl and 3 mM MgCl2 (all Life Technologies, final concentrations are shown) were added to a final volume of 10 μl.

    Affinity Magnetic Separation:

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    Reverse Transcription Polymerase Chain Reaction:

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    Immunofluorescence:

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    Molecular Weight:

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    Nucleic Acid Electrophoresis:

    Article Title: An Immunoreactive 38-Kilodalton Protein of Ehrlichia canis Shares Structural Homology and Iron-Binding Capacity with the Ferric Ion-Binding Protein Family
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    Multiple Displacement Amplification:

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    Isolation:

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    Article Snippet: After phase separation with chloroform, the aqueous fraction was mixed with an equal volume of 100% ethanol and subjected to RNA isolation using a Direct-Zol RNA miniprep kit (Zymo Research) according to the manufacturers' recommendations, with on-column DNase 1 treatment. .. Subsequently, the RT master mix containing 4 μl of 5x first strand buffer, 1 μl 0.1 M dithiothreitol, 1 μl dNTP mix (10 mM each), 1 μl Superscript IV reverse transcriptase (200 U/μl, Invitrogen), 1 μl RNasin Plus RNase inhibitor (40 U/μl, Promega) was added and reactions were incubated at 60°C for 10 minutes followed by heating at 80°C for 10 minutes.

    Microscopy:

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    Article Snippet: Microscopy was performed on a Zeiss LSM 510 META confocal microscope. .. Bloodstream-form cells (5 × 106 ) were lysed in SDS-sample buffer, 0.1 M dithiothreitol and run on a 4–12% Nupage gel with MOPS running buffer (Invitrogen), transferred to nitrocellulose and developed using 1:1000 mouse anti-TbPMI, 1:100 000 anti-mouse horseradish peroxidase conjugate (Stratech, UK) and West Pico ECL (Pierce/ThermoScientific) and Hyperfilm ECL film (GE Healthcare, UK).

    Purification:

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    Article Title: Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer
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    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase
    Article Snippet: After cleavage with trifluoroacetic acid, peptide purification was performed on a reverse-phase HPLC with Shim-pack C18 column equipment (Shimadzu Corp.). .. The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    Dot Blot:

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    Article Snippet: .. Dot blot analyses were conducted with either native protein, protein plus 0.1% (wt/vol) SDS, protein with 50 mM dithiothreitol (Invitrogen), or a combination of SDS plus the reducing agent. ..

    Article Title: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis
    Article Snippet: The samples were treated with or without reducing agent 100 mM dithiothreitol (DTT) (Thermo Fisher Scientific, R0861). (ii) SOD1 aggregate formation was verified by filter trap assay as detailed in [ ]. .. Briefly, 1 μg/μl protein NSC34 cell extracts treated with or without DTT were filtrated through a 0.2 μm cellulose acetate membrane by using a BRL dot-blot filtration unit.

    Polymerase Chain Reaction:

    Article Title: Properties of targeted preamplification in DNA and cDNA quantification
    Article Snippet: .. Preamplification additives The effects of 18 different PCR additives on the preamplification reaction were evaluated (final tested concentrations are shown): 7-deaza-2′-deoxyguanosine 5′-triphosphate lithium salt (50 and 100 µM; Sigma-Aldrich), ammonium sulfate (10 and 15 mM; Sigma-Aldrich), betaine (0.25 and 0.50 M; Sigma-Aldrich), bovine serum albumin supplied at 20 mg/ml in 10 mM Tris-HCl, 100 mM KCl, 1 mM EDTA and 50% glycerol (1 and 2 µg/µl; Thermo Scientific), D-(+)-trehalose dehydrate (0.15 and 0.30 M; Sigma-Aldrich), dimethyl sulfoxide (1%; Sigma-Aldrich), dithiothreitol (1.5 and 3.0 mM; Life Technologies), formamide (0.5 M; Sigma-Aldrich), gelatin (0.01 and 0.10%; Sigma-Aldrich), glycerol (2.5 and 5.0%; Sigma-Aldrich), IGEPAL CA-630 (0.25 and 0.50%, Sigma-Aldrich), l -carnitine inner salt (0.25 and 0.50 M; Sigma-Aldrich), GenElute-LPA (50 and 100 ng/µl; Sigma-Aldrich), polyinosinic:polycytidylic acid potassium salt (5 and 50 ng/µl; Sigma-Aldrich), tetramethylammonium chloride (30 and 60 mM; Sigma-Aldrich), Triton X-100 (0.2 and 0.4%; Sigma-Aldrich), TWEEN 20 (0.10 and 0.50%, Sigma-Aldrich) and yeast tRNA (50 and 100 ng/µl, Life Technologies). .. High-throughput quantitative real-time PCR High-throughput qPCR was performed on the BioMark system using the 96.96 Dynamic Array Chip for Gene Expression and EvaGreen-based detection.

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection
    Article Snippet: For strand-specific PCR (Figs , , , and A Fig), RT primers that distinguish the EBOV gRNA, cRNA and VP40 mRNA were designed with a unique 18–20 nucleotide tag (highlighted in bold) adjacent to the strand specific sequence [ ]. .. Subsequently, the RT master mix containing 4 μl of 5x first strand buffer, 1 μl 0.1 M dithiothreitol, 1 μl dNTP mix (10 mM each), 1 μl Superscript IV reverse transcriptase (200 U/μl, Invitrogen), 1 μl RNasin Plus RNase inhibitor (40 U/μl, Promega) was added and reactions were incubated at 60°C for 10 minutes followed by heating at 80°C for 10 minutes.

    Protein Extraction:

    Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells
    Article Snippet: The membrane fraction from monocytes stimulated by rsCD26-coated polystyrene beads was extracted with a ReadyPrep protein extraction kit (Bio-Rad) according to the manufacturer's instructions. .. Membrane proteins were then cleaned up to pellets with a two-dimensional (2D) Clean-Up kit (Bio-Rad) and resuspended in rehydration lysis buffer {8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range, 3 to 10) (Invitrogen), 0.002% bromphenol blue} to a final concentration of 1 mg/ml.

    Confocal Microscopy:

    Article Title: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis
    Article Snippet: The samples were treated with or without reducing agent 100 mM dithiothreitol (DTT) (Thermo Fisher Scientific, R0861). (ii) SOD1 aggregate formation was verified by filter trap assay as detailed in [ ]. .. SOD1 aggregates were detected with anti-SOD1 antibody, following the immunological protocol described above. (iii) SOD1 inclusions were monitored by confocal microscopy.

    Lysis:

    Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells
    Article Snippet: .. Membrane proteins were then cleaned up to pellets with a two-dimensional (2D) Clean-Up kit (Bio-Rad) and resuspended in rehydration lysis buffer {8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range, 3 to 10) (Invitrogen), 0.002% bromphenol blue} to a final concentration of 1 mg/ml. .. Wide-range immobilized pH gradient strips (pH 3-10NL; Invitrogen) were rehydrated in 155 μl of rehydration lysis buffer containing 50 μg protein and isoelectric focused at 1,367 V · h on a ZOOM IPGRunner system (Invitrogen).

    SDS Page:

    Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells
    Article Snippet: Membrane proteins were then cleaned up to pellets with a two-dimensional (2D) Clean-Up kit (Bio-Rad) and resuspended in rehydration lysis buffer {8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range, 3 to 10) (Invitrogen), 0.002% bromphenol blue} to a final concentration of 1 mg/ml. .. Second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4 to 12% NuPAGE bis-Tris gels (Invitrogen).

    Article Title: Arctic Micromonas uses protein pools and non-photochemical quenching to cope with temperature restrictions on Photosystem II protein turnover
    Article Snippet: Dithiothreitol was added to a final concentration of 50 mM with the remaining sample volume made up with 1× Bolt sample buffer from Life Technologies. .. Proteins were separated by SDS-PAGE electrophoresis in 4–12 % Bis Tris Plus 17 well polyacrylamide gels from Life Technologies.

    Article Title: Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells
    Article Snippet: .. SDS-PAGE for tryptic digestion Samples were boiled in 2% SDS-loading buffer with dithiothreitol and subjected to SDS-PAGE using a NuPAGE Novex Bis-Tris Mini gel (4–12%) with NuPAGE MOPS SDS-Running buffer supplemented with 1× NuPAGE antioxidant (Life Technologies). .. After electrophoresis, the gel was stained with InstantBlue protein stain and cut into approximately 2 × 6 mm slices then each slice further cut into 1 mm2 pieces.

    Article Title: Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulfide bond formation to an Escherichia coli strain lacking DsbA
    Article Snippet: For reduction of disulfide bonds, TCA-precipitated proteins were incubated in 100 μl 100 mM Tris.HCl, pH 8.0 containing 0.1% SDS and 100 mM dithiothreitol (DTT, Invitrogen) for 30 min at room temperature. .. Non-reducing 3X-SDS sample buffer (40 μl) was then added to each sample, heated for 10 min at 100 °C and 15 μl of sample was applied to SDS-PAGE directly.

    Real-time Polymerase Chain Reaction:

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection
    Article Snippet: Subsequently, the RT master mix containing 4 μl of 5x first strand buffer, 1 μl 0.1 M dithiothreitol, 1 μl dNTP mix (10 mM each), 1 μl Superscript IV reverse transcriptase (200 U/μl, Invitrogen), 1 μl RNasin Plus RNase inhibitor (40 U/μl, Promega) was added and reactions were incubated at 60°C for 10 minutes followed by heating at 80°C for 10 minutes. .. The unique tag sequence (bold capital letters) used in the RT primers were used as forward primers in the strand-specific qPCR, whereas both probes and reverse primers were specific to the EBOV genome (capital letters).

    RNA Extraction:

    Article Title: Properties of targeted preamplification in DNA and cDNA quantification
    Article Snippet: Paragraph title: RNA extraction & reverse transcription ... 100 U SuperScript III, 20 U RNaseOUT, 5 mM dithiothreitol, 50 mM Tris-HCl, pH 8.3, 75 mM KCl and 3 mM MgCl2 (all Life Technologies, final concentrations are shown) were added to a final volume of 10 μl.

    Concentration Assay:

    Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells
    Article Snippet: .. Membrane proteins were then cleaned up to pellets with a two-dimensional (2D) Clean-Up kit (Bio-Rad) and resuspended in rehydration lysis buffer {8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range, 3 to 10) (Invitrogen), 0.002% bromphenol blue} to a final concentration of 1 mg/ml. .. Wide-range immobilized pH gradient strips (pH 3-10NL; Invitrogen) were rehydrated in 155 μl of rehydration lysis buffer containing 50 μg protein and isoelectric focused at 1,367 V · h on a ZOOM IPGRunner system (Invitrogen).

    Article Title: Arctic Micromonas uses protein pools and non-photochemical quenching to cope with temperature restrictions on Photosystem II protein turnover
    Article Snippet: .. Dithiothreitol was added to a final concentration of 50 mM with the remaining sample volume made up with 1× Bolt sample buffer from Life Technologies. .. Standards were made by diluting stock PsbA or FtsH protein standard in the same Bolt sample buffer with a final concentration of 50 mM dithiothreitol.

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection
    Article Snippet: The concentration of the extracted RNA was measured with the NanoDrop 2000 (ThermoFisher Scientific) and adjusted to 100 ng/μl. .. Subsequently, the RT master mix containing 4 μl of 5x first strand buffer, 1 μl 0.1 M dithiothreitol, 1 μl dNTP mix (10 mM each), 1 μl Superscript IV reverse transcriptase (200 U/μl, Invitrogen), 1 μl RNasin Plus RNase inhibitor (40 U/μl, Promega) was added and reactions were incubated at 60°C for 10 minutes followed by heating at 80°C for 10 minutes.

    Article Title: Properties of targeted preamplification in DNA and cDNA quantification
    Article Snippet: 100 U SuperScript III, 20 U RNaseOUT, 5 mM dithiothreitol, 50 mM Tris-HCl, pH 8.3, 75 mM KCl and 3 mM MgCl2 (all Life Technologies, final concentrations are shown) were added to a final volume of 10 μl. .. The following temperature profile was applied: 25°C for 5 min, 50°C for 60 min, 55°C for 15 min and 70°C for 15 min. cDNA was diluted to a concentration corresponding to 1 ng/μl with nuclease-free water and stored at –20°C.

    Article Title: Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer
    Article Snippet: Materials Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (Sulfo-LC-SPDP), and dithiothreitol were purchased from Pierce Biotechnology (Rockford IL, USA). .. Seventy percent concentrated nitric acid (3-aminopropyl) triethoxysilane (APTES), and all reagents for spectrophotometric determination of iron concentration, including hydroquinone, o-phenanthroline, and ammonium iron (II) sulfate hexahydrate, were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Staining:

    Article Title: CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells
    Article Snippet: Membrane proteins were then cleaned up to pellets with a two-dimensional (2D) Clean-Up kit (Bio-Rad) and resuspended in rehydration lysis buffer {8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range, 3 to 10) (Invitrogen), 0.002% bromphenol blue} to a final concentration of 1 mg/ml. .. Analytical gels were stained with colloidal Coomassie brilliant blue R250.

    Article Title: Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells
    Article Snippet: SDS-PAGE for tryptic digestion Samples were boiled in 2% SDS-loading buffer with dithiothreitol and subjected to SDS-PAGE using a NuPAGE Novex Bis-Tris Mini gel (4–12%) with NuPAGE MOPS SDS-Running buffer supplemented with 1× NuPAGE antioxidant (Life Technologies). .. After electrophoresis, the gel was stained with InstantBlue protein stain and cut into approximately 2 × 6 mm slices then each slice further cut into 1 mm2 pieces.

    Fluorescence In Situ Hybridization:

    Article Title: The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei
    Article Snippet: Samples were then blocked in 5% fish skin gelatin (FSG) in PBS containing 10% normal goat serum. .. Bloodstream-form cells (5 × 106 ) were lysed in SDS-sample buffer, 0.1 M dithiothreitol and run on a 4–12% Nupage gel with MOPS running buffer (Invitrogen), transferred to nitrocellulose and developed using 1:1000 mouse anti-TbPMI, 1:100 000 anti-mouse horseradish peroxidase conjugate (Stratech, UK) and West Pico ECL (Pierce/ThermoScientific) and Hyperfilm ECL film (GE Healthcare, UK).

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  • 95
    Thermo Fisher m dithiothreitol
    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) <t>dithiothreitol.</t> Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.
    M Dithiothreitol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher m dtt
    Polysulfides inhibit PTEN by sulfane sulfur addition. (A) Recombinant PTEN-WT was <t>prereduced</t> with 20 m M <t>DTT</t> for 10 min, followed by buffer exchange into a nonreducing PTEN assay buffer and exposure to 100 μ M H 2 O 2 or NaHS
    M Dtt, supplied by Thermo Fisher, 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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    88
    Thermo Fisher u2os cell lysate
    HuR co-immunoprecipitation with Bcl-2 protein. A , <t>U2OS</t> cells were transfected with FLAG or with FLAG-Bcl2 expressing plasmid. 48 h later, cell lysates from transfected and untransfected cells were immunoprecipitated with anti-FLAG-incubated A/G resin.
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    93
    Thermo Fisher gst lysis buffer
    Disruption of TFIIB autoinhibition by Ssu72. A , competition cross-linking between SUMO <t>TFIIBn</t> W52pBpA and Ssu72 for TFIIBc WT visualized by anti-FLAG Western blotting. Lanes labeled Mut utilized the TFIIBc K189E/R193E mutant. Bottom , TFIIBc-FLAG becomes distorted in some lanes due to <t>GST</t> that runs in a position similar to that of TFIIBc. B , competition cross-linking assay between Ssu72 C131pBpA and either GST-TFIIBn or GST-VP16AD for TFIIBc as visualized by FLAG antibody Western blotting.
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    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Redox Regulation of Methionine Aminopeptidase 2 Activity *

    doi: 10.1074/jbc.M114.554253

    Figure Lengend Snippet: Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.

    Article Snippet: Just before use, the active site disulfide bond of the oxidoreductase was reduced with 50 m m dithiothreitol and 50 m m tris-(2-carboxyethyl)phosphine (TCEP) for 1 h at 25 °C, and the reducing agents were removed by two passes through Zeba spin desalting columns equilibrated with phosphate-buffered saline (Thermo Scientific).

    Techniques: Labeling, SDS Page

    Polysulfides inhibit PTEN by sulfane sulfur addition. (A) Recombinant PTEN-WT was prereduced with 20 m M DTT for 10 min, followed by buffer exchange into a nonreducing PTEN assay buffer and exposure to 100 μ M H 2 O 2 or NaHS

    Journal: Antioxidants & Redox Signaling

    Article Title: Polysulfides Link H2S to Protein Thiol Oxidation

    doi: 10.1089/ars.2012.5041

    Figure Lengend Snippet: Polysulfides inhibit PTEN by sulfane sulfur addition. (A) Recombinant PTEN-WT was prereduced with 20 m M DTT for 10 min, followed by buffer exchange into a nonreducing PTEN assay buffer and exposure to 100 μ M H 2 O 2 or NaHS

    Article Snippet: Typically, roGFP2-His was prereduced with 20 m M DTT for 10 min before the experiment, and passed through gel filtration columns (Thermo Scientific) pre-equilibrated with the roGFP2 assay buffer to remove DTT immediately before the start of the assay.

    Techniques: Recombinant, Buffer Exchange

    HuR co-immunoprecipitation with Bcl-2 protein. A , U2OS cells were transfected with FLAG or with FLAG-Bcl2 expressing plasmid. 48 h later, cell lysates from transfected and untransfected cells were immunoprecipitated with anti-FLAG-incubated A/G resin.

    Journal: The Journal of Biological Chemistry

    Article Title: B Cell Lymphoma (Bcl)-2 Protein Is the Major Determinant in bcl-2 Adenine-Uridine-rich Element Turnover Overcoming HuR Activity *

    doi: 10.1074/jbc.M109.023721

    Figure Lengend Snippet: HuR co-immunoprecipitation with Bcl-2 protein. A , U2OS cells were transfected with FLAG or with FLAG-Bcl2 expressing plasmid. 48 h later, cell lysates from transfected and untransfected cells were immunoprecipitated with anti-FLAG-incubated A/G resin.

    Article Snippet: For FLAG IP assays, 0.5 ml of U2OS cell lysate (10 m m Hepes, pH 7.6, 100 m m KCl, 5 m m MgCl2 , 2.5 m m EDTA, 2 m m dithiothreitol, protease, and RNase inhibitors) were incubated for 2 h at 4 °C with protein A/G UltraLink Resin (Thermo Fisher Scientific), precoated with 2.0 μg of FLAG M2 mouse monoclonal antibody (Sigma).

    Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Incubation

    Disruption of TFIIB autoinhibition by Ssu72. A , competition cross-linking between SUMO TFIIBn W52pBpA and Ssu72 for TFIIBc WT visualized by anti-FLAG Western blotting. Lanes labeled Mut utilized the TFIIBc K189E/R193E mutant. Bottom , TFIIBc-FLAG becomes distorted in some lanes due to GST that runs in a position similar to that of TFIIBc. B , competition cross-linking assay between Ssu72 C131pBpA and either GST-TFIIBn or GST-VP16AD for TFIIBc as visualized by FLAG antibody Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Structural dissection of an interaction between transcription initiation and termination factors implicated in promoter-terminator cross-talk

    doi: 10.1074/jbc.M117.811521

    Figure Lengend Snippet: Disruption of TFIIB autoinhibition by Ssu72. A , competition cross-linking between SUMO TFIIBn W52pBpA and Ssu72 for TFIIBc WT visualized by anti-FLAG Western blotting. Lanes labeled Mut utilized the TFIIBc K189E/R193E mutant. Bottom , TFIIBc-FLAG becomes distorted in some lanes due to GST that runs in a position similar to that of TFIIBc. B , competition cross-linking assay between Ssu72 C131pBpA and either GST-TFIIBn or GST-VP16AD for TFIIBc as visualized by FLAG antibody Western blotting.

    Article Snippet: Cells containing GST fusion proteins (TFIIBn and VP16AD) were lysed by sonication in GST lysis buffer (50 m m Tris, pH 8.0, 150 m m NaCl, and 5 m m DTT), and clarified lysate was bound to glutathione-agarose (Thermo Scientific).

    Techniques: Western Blot, Labeling, Mutagenesis