dithiothreitol  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dithiothreitol
    Dithiothreitol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol/product/Thermo Fisher
    Average 99 stars, based on 107 article reviews
    Price from $9.99 to $1999.99
    dithiothreitol - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response
    Article Snippet: MEF cells (5 × 107 cells) were suspended in a homogenization buffer (10 mM Hepes-KOH, pH 7.5, 10 mM KoAc, 1.5 mM Mg(OAc)2 , 2 mM dithiothreitol, 1 mM PMSF, and 200 U/ml RNaseOUT ribonuclease inhibitor [Thermo Fisher Scientific]), then ruptured by a Dounce homogenizer. .. To separate microsomal and cytosolic fractions, homogenates were subjected to low-speed centrifugation (1,500 ×g ) for 5 min.

    Amplification:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 . .. The purified cDNA was click-ligated with Illumina adapters and final PCR amplification with indexes.

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr. .. Subsequently, two rounds of PCR amplification with nested primer pairs were performed; the template for the second PCR was 1 μl of the first reaction.

    Filtration:

    Article Title: Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody
    Article Snippet: The guanidine HCl (24115) used for reduced mass analysis, trifluoroacetic acid (TFA, 28904), and dithiothreitol (DTT, 20290) were from Thermo Scientific. .. Sephadex G-25-packed NAP-5 gel filtration columns were from GE Life Sciences (17085301).

    Positive Control:

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: As a positive control, MS2 bacteriophage RNA was spiked into a background of PAO1 RNA at 100 copies per cell equivalent and used as a tester against PAO1. .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL).

    Synthesized:

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL). .. The second strand was synthesized in a 150-μl polymerization reaction mixture containing 1× second-strand buffer [20 mM Tris-HCl (pH 6.9), 4.6 mM MgCl2 , 90 mM KCl, 0.15 mM β-NAD+ , 10 mM (NH4 )2 SO4 ], 0.2 mM dNTPs, 40 U of E. coli DNA polymerase I, 10 U of E. coli DNA ligase, 2 U of ribonuclease H (all enzymes obtained from Gibco-BRL).

    Quantitative RT-PCR:

    Article Title: Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... For cDNA synthesis, 20 µl 5X Moloney murine leukemia virus (MMLV) buffer [containing 250 mmol/l Tris-HCl (pH 8.3), 375 mmol/l KCl, and 15 mmol/l MgCl2 ; Thermo Fisher Scientific, Inc.], 10 µl dithiothreitol (100 mmol/l; Thermo Fisher Scientific, Inc.), 10 µl dNTP (each 10 mmol/l; Amersham Pharmacia Biotech), 0.5 µl random hexamers [50 OD dissolved in 550 µl of 10 mmol/l Tris-HCl (pH 7.5), and 1 mmol/l EDTA; Amersham Pharmacia Biotech, Piscataway, NJ, USA], 2.5 µl bovine serum albumin [3 mg/ml in 10 mmol/l Tris-HC1 (pH 7.5); Amersham Pharmacia Biotech], 2.5 µl RNAse inhibitor (5 × 1,000 units; Amersham Pharmacia Biotech), and 5 µl MMLV reverse transcriptase (200 U/µl; Thermo Fisher Scientific, Inc.), added to a total volume of 50.5 µl.

    Incubation:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: After 5 min incubation at 30°C, reaction was quenched on ice for 5 min with 2 volumes of 10 mM Tris pH 7.4 and 30% 2-mercaptoethanol (BME). .. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 .

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: .. The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr. .. Subsequently, two rounds of PCR amplification with nested primer pairs were performed; the template for the second PCR was 1 μl of the first reaction.

    Article Title: AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability
    Article Snippet: After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl. .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl.

    Activity Assay:

    Article Title: AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability
    Article Snippet: Activity coefficients were calculated from the Debye–Hückel equation ( ) to be 0.718 for Cs+ and 0.416 for Mg2+ in the intracellular solution, 0.496 for Ca2+ in the 15 m m Ca2+ solution, and 0.357 for Ca2+ in the 50 m m Ca2+ solution. .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl.

    Expressing:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: .. For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: The protocol described by Lisitsyn et al. ( ) was modified for use with bacterial cDNA as a means of analyzing gene expression. .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL).

    Modification:

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: The protocol described by Lisitsyn et al. ( ) was modified for use with bacterial cDNA as a means of analyzing gene expression. .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL).

    High Performance Liquid Chromatography:

    Article Title: Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody
    Article Snippet: The guanidine hydrochloride (G9284) and tris base (T6791) used for peptide mapping, along with the EDTA (E4884), Chromasolv HPLC-grade 2-Propanol (IPA, 34863), iodoacetamide (I6125), and calcium chloride (C5670) were from Sigma Aldrich. .. The guanidine HCl (24115) used for reduced mass analysis, trifluoroacetic acid (TFA, 28904), and dithiothreitol (DTT, 20290) were from Thermo Scientific.

    Silver Staining:

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample. .. Protein bands were visualized using silver staining, and band density was measured using ImageJ analysis software [ , ].

    Dissection:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. For pulmonary tissue protein expression analysis, mouse pups were euthanized using 200 mg/kg pentobarbital sodium intraperitoneal injection, and whole lung tissues were obtained by dissection and frozen in liquid N2 .

    Indirect Immunoperoxidase Assay:

    Article Title: Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody
    Article Snippet: The guanidine hydrochloride (G9284) and tris base (T6791) used for peptide mapping, along with the EDTA (E4884), Chromasolv HPLC-grade 2-Propanol (IPA, 34863), iodoacetamide (I6125), and calcium chloride (C5670) were from Sigma Aldrich. .. The guanidine HCl (24115) used for reduced mass analysis, trifluoroacetic acid (TFA, 28904), and dithiothreitol (DTT, 20290) were from Thermo Scientific.

    Polymerase Chain Reaction:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 . .. The purified cDNA was click-ligated with Illumina adapters and final PCR amplification with indexes.

    Article Title: Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... For cDNA synthesis, 20 µl 5X Moloney murine leukemia virus (MMLV) buffer [containing 250 mmol/l Tris-HCl (pH 8.3), 375 mmol/l KCl, and 15 mmol/l MgCl2 ; Thermo Fisher Scientific, Inc.], 10 µl dithiothreitol (100 mmol/l; Thermo Fisher Scientific, Inc.), 10 µl dNTP (each 10 mmol/l; Amersham Pharmacia Biotech), 0.5 µl random hexamers [50 OD dissolved in 550 µl of 10 mmol/l Tris-HCl (pH 7.5), and 1 mmol/l EDTA; Amersham Pharmacia Biotech, Piscataway, NJ, USA], 2.5 µl bovine serum albumin [3 mg/ml in 10 mmol/l Tris-HC1 (pH 7.5); Amersham Pharmacia Biotech], 2.5 µl RNAse inhibitor (5 × 1,000 units; Amersham Pharmacia Biotech), and 5 µl MMLV reverse transcriptase (200 U/µl; Thermo Fisher Scientific, Inc.), added to a total volume of 50.5 µl.

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: Paragraph title: Reverse transcriptase PCR analysis ... Reverse transcription on 1 μg of total RNA was performed at 50°C for 1 h with 200 ng of random primers, 0.2 mM dNTPs, 1× First-Strand Buffer, 5 mM dithiothreitol, and 200 U of SuperScript III Reverse Transcriptase (all from ThermoFisher Scientific).

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr. .. Subsequently, two rounds of PCR amplification with nested primer pairs were performed; the template for the second PCR was 1 μl of the first reaction.

    Article Title: AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability
    Article Snippet: After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl. .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl.

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL). .. All adapter sequences and PCR conditions were as described by Lisitsyn et al. , with exceptions as noted.

    Isolation:

    Article Title: Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response
    Article Snippet: Paragraph title: Isolation of subcellular fractions ... MEF cells (5 × 107 cells) were suspended in a homogenization buffer (10 mM Hepes-KOH, pH 7.5, 10 mM KoAc, 1.5 mM Mg(OAc)2 , 2 mM dithiothreitol, 1 mM PMSF, and 200 U/ml RNaseOUT ribonuclease inhibitor [Thermo Fisher Scientific]), then ruptured by a Dounce homogenizer.

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: Total bacterial RNA (3 μg), isolated from mid-log-phase cultures, was DNase treated and converted to double-stranded cDNA. .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL).

    Sequencing:

    Article Title: Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines
    Article Snippet: For cDNA synthesis, 20 µl 5X Moloney murine leukemia virus (MMLV) buffer [containing 250 mmol/l Tris-HCl (pH 8.3), 375 mmol/l KCl, and 15 mmol/l MgCl2 ; Thermo Fisher Scientific, Inc.], 10 µl dithiothreitol (100 mmol/l; Thermo Fisher Scientific, Inc.), 10 µl dNTP (each 10 mmol/l; Amersham Pharmacia Biotech), 0.5 µl random hexamers [50 OD dissolved in 550 µl of 10 mmol/l Tris-HCl (pH 7.5), and 1 mmol/l EDTA; Amersham Pharmacia Biotech, Piscataway, NJ, USA], 2.5 µl bovine serum albumin [3 mg/ml in 10 mmol/l Tris-HC1 (pH 7.5); Amersham Pharmacia Biotech], 2.5 µl RNAse inhibitor (5 × 1,000 units; Amersham Pharmacia Biotech), and 5 µl MMLV reverse transcriptase (200 U/µl; Thermo Fisher Scientific, Inc.), added to a total volume of 50.5 µl. .. Quantification of six genes of interest and an internal reference gene encoding β-actin was performed using the fluorescence-based real-time detection method by a ABI PRISM 7900 Sequence detection system (Perkin-Elmer Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr. .. The large sequence diversity of K+ channel subunits at the nucleotide level required the use of multiple sets of specific primers rather than a single set of degenerate primers.

    Sonication:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    Injection:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. For pulmonary tissue protein expression analysis, mouse pups were euthanized using 200 mg/kg pentobarbital sodium intraperitoneal injection, and whole lung tissues were obtained by dissection and frozen in liquid N2 .

    Nucleic Acid Electrophoresis:

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample. .. Gel electrophoresis was performed within a Bio-Rad Mini-PROTEAN® Tetra Cell (Bio-Rad, cat. no. 1658004) at 200 V for 25 min in a TGX running buffer (25 mM tris-base, 192 mM glycine, and 0.1% SDS, pH = 8.3).

    In Vivo:

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: Paragraph title: In vivo EDC probing of rice ... For treatments using a DTT quench, 1 g of DL-1,4 dithiothreitol (Acros Organics; 16568_0250) was added to the tube, which was then shaken vigorously for 2 min. Then, the reaction buffer was decanted and the seedlings were washed three times with ∼20 mL deionized water for each wash before immediate drying and quick freezing in liquid N2 .

    Fluorescence:

    Article Title: Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines
    Article Snippet: For cDNA synthesis, 20 µl 5X Moloney murine leukemia virus (MMLV) buffer [containing 250 mmol/l Tris-HCl (pH 8.3), 375 mmol/l KCl, and 15 mmol/l MgCl2 ; Thermo Fisher Scientific, Inc.], 10 µl dithiothreitol (100 mmol/l; Thermo Fisher Scientific, Inc.), 10 µl dNTP (each 10 mmol/l; Amersham Pharmacia Biotech), 0.5 µl random hexamers [50 OD dissolved in 550 µl of 10 mmol/l Tris-HCl (pH 7.5), and 1 mmol/l EDTA; Amersham Pharmacia Biotech, Piscataway, NJ, USA], 2.5 µl bovine serum albumin [3 mg/ml in 10 mmol/l Tris-HC1 (pH 7.5); Amersham Pharmacia Biotech], 2.5 µl RNAse inhibitor (5 × 1,000 units; Amersham Pharmacia Biotech), and 5 µl MMLV reverse transcriptase (200 U/µl; Thermo Fisher Scientific, Inc.), added to a total volume of 50.5 µl. .. Quantification of six genes of interest and an internal reference gene encoding β-actin was performed using the fluorescence-based real-time detection method by a ABI PRISM 7900 Sequence detection system (Perkin-Elmer Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Methylation:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: Methylated FHV RNAs and respective controls were processed with ClickSeq library construction method as describe above with the exception of the use of the high-fidelity and processive thermostable group II reverse transcriptase enzyme (TGIRT-III, InGex) during reverse transcription. .. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 .

    Random Primed:

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: .. The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL). .. The second strand was synthesized in a 150-μl polymerization reaction mixture containing 1× second-strand buffer [20 mM Tris-HCl (pH 6.9), 4.6 mM MgCl2 , 90 mM KCl, 0.15 mM β-NAD+ , 10 mM (NH4 )2 SO4 ], 0.2 mM dNTPs, 40 U of E. coli DNA polymerase I, 10 U of E. coli DNA ligase, 2 U of ribonuclease H (all enzymes obtained from Gibco-BRL).

    Transferring:

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: The cytoplasm of a whole-cell recorded neuron, either including (Kv2, Kv3, and Kv4) or excluding (Kv1) the nucleus, was harvested into the patch pipette by applying negative pressure. .. The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr.

    Article Title: AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability
    Article Snippet: .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl. .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl.

    Size-exclusion Chromatography:

    Article Title: Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines
    Article Snippet: The supernatant was then carefully poured off, so as not to disturb the RNA pellet, and the samples were quick-spun for 15 sec at 13,000 × g. The remaining ethanol was removed, and the samples were left to air-dry for 15 min. .. For cDNA synthesis, 20 µl 5X Moloney murine leukemia virus (MMLV) buffer [containing 250 mmol/l Tris-HCl (pH 8.3), 375 mmol/l KCl, and 15 mmol/l MgCl2 ; Thermo Fisher Scientific, Inc.], 10 µl dithiothreitol (100 mmol/l; Thermo Fisher Scientific, Inc.), 10 µl dNTP (each 10 mmol/l; Amersham Pharmacia Biotech), 0.5 µl random hexamers [50 OD dissolved in 550 µl of 10 mmol/l Tris-HCl (pH 7.5), and 1 mmol/l EDTA; Amersham Pharmacia Biotech, Piscataway, NJ, USA], 2.5 µl bovine serum albumin [3 mg/ml in 10 mmol/l Tris-HC1 (pH 7.5); Amersham Pharmacia Biotech], 2.5 µl RNAse inhibitor (5 × 1,000 units; Amersham Pharmacia Biotech), and 5 µl MMLV reverse transcriptase (200 U/µl; Thermo Fisher Scientific, Inc.), added to a total volume of 50.5 µl.

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: SEC was performed on an NGC Bio-RAD equipped with one Bio-RAD ENrich 650 (cat. no. 780-1650) and two Bio-RAD ENrich 70 columns (cat. no. 780-1070) linked in series. .. Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Functional and Molecular Differences between Voltage-Gated K+ Channels of Fast-Spiking Interneurons and Pyramidal Neurons of Rat Hippocampus
    Article Snippet: Patch pipettes used for RT-PCR experiments (0.8–3 MΩ) were filled with autoclaved internal solution containing (in m m ): 140 KCl, 5 EGTA, 3 MgCl2 , and 5 HEPES, pH-adjusted to 7.3 with KOH. .. The harvested material was then expelled under visual control into an autoclaved reaction tube containing hexamer random primers, deoxyribonucleoside triphosphates, dithiothreitol, ribonuclease inhibitor, and Superscript reverse transcriptase II (Life Technologies, Gaithersburg, MD), and incubated at 37°C for 1 hr.

    Article Title: AMPA Receptor Calcium Permeability, GluR2 Expression, and Selective Motoneuron Vulnerability
    Article Snippet: Single-cell RT-PCR and restriction analysis. .. After electrophysiological recording the cell content was aspirated into the patch pipette and expelled with the pipette solution (4 μl) into the reverse transcription mix containing: 5× first-strand buffer (2.5 μl; Life Technologies), dithiothreitol (1 μl; Life Technologies), dNTPs (4 μl of a 2.5 m m stock; Amersham Pharmacia Biotech, Piscataway, NJ), random hexamers (1 μl of a 2.5 μg/μl stock; Boehringer Mannheim, Indianapolis, IN), RNase inhibitor (0.5 μl or 20 U; Promega, Madison, WI), and reverse transcriptase (Life Technologies Superscript II; 0.5 μl or 100 U) to a total of 13.5 μl.

    Staining:

    Article Title: Evolutionary compaction and adaptation visualized by the structure of the dormant microsporidian ribosome
    Article Snippet: Ribosome preparations were analysed for homogeneity by negative stain EM, and cryo-EM grids were prepared from the most homogeneous sample (see , ). .. Then, 3.5 μl of sample in buffer A (30 mM Tris-HCl, pH 7.5 (4°C), 5 mM magnesium acetate, 25 mM KC1, 1 mM dithiothreitol and 1 mM ethylenediaminetetraacetic acid) at an absorbance of 11 mAU (AU is arbitrary units) at 260 nm (Nanodrop 2000, Thermo Scientific) were applied to glow-discharged (30 s at 50 mA), lacy-carbon grids (TED PELLA, Inc., product no. 01824) and flash frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 100% humidity, blot force of 0 and blot time 2.5 s.

    Purification:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: In this study, nuclease-treated and purified FHV virions were supplemented with DMS to 5% final concentration. .. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 .

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: Following oxidative cross-linking, the tubes were stored at −20 °C pending purification via SEC. Analytical size exclusion chromatography (SEC) of cross-linked oligomers. .. Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample.

    Article Title: Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability
    Article Snippet: The first strand of cDNA was synthesized in a 20-μl random-primed reverse transcription reaction mixture containing 1× first-strand buffer (50 mM Tris-HCl [pH 8.3], 40 mM KCl, 6 mM MgCl2 ; Gibco-BRL), 100 ng of primer [2:1:1 mix of N6 (SN)3 , and (NS)3 , where S = G or C] 0.5 mM deoxynucleoside triphosphates (dNTPs), 10 mM dithiothreitol, 5% dimethyl sulfoxide (DMSO), and 200 U of reverse transcriptase (SuperScript II; Gibco-BRL). .. Purified cDNA was digested with Dpn II and ligated with oligonucleotide (R-Bgl) adapters.

    SDS Page:

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: Protein mixtures were separated via SDS-PAGE using 12% Mini-PROTEAN® TGX™ polyacrylamide gels (Bio-Rad, cat. no. 4561044). .. Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample.

    Software:

    Article Title: Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures
    Article Snippet: Then, 10 µL of 6X Laemmli SDS sample buffer containing 20 mM dithiothreitol (Alfa Aesar, cat. no. J60660, Haverhill, MA, USA) was added to 50 µL of each sample. .. Protein bands were visualized using silver staining, and band density was measured using ImageJ analysis software [ , ].

    RNA Extraction:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: RNA extraction was conducted with Quick-RNA Viral Kits (Zymo Research) with additional BME in the extraction buffer. .. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 .

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: For treatments using a DTT quench, 1 g of DL-1,4 dithiothreitol (Acros Organics; 16568_0250) was added to the tube, which was then shaken vigorously for 2 min. Then, the reaction buffer was decanted and the seedlings were washed three times with ∼20 mL deionized water for each wash before immediate drying and quick freezing in liquid N2 . .. Frozen seedlings then were subjected to total RNA extraction as described below, with separate mortars and pestles used for each treatment.

    In Vitro:

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: Paragraph title: In vitro EDC probing of rice RNA ... For reactions testing a dithiothreitol (DTT) quench, three separate quench solutions were prepared: DL-1,4 dithiothreitol (Acros Organics; 16568_0250) dissolved to 2.5 M in deionized water; 1 g of DTT dissolved in 5 mL of 1 M sodium acetate (pH 5); or 1 M sodium acetate (pH 5).

    Homogenization:

    Article Title: Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response
    Article Snippet: .. MEF cells (5 × 107 cells) were suspended in a homogenization buffer (10 mM Hepes-KOH, pH 7.5, 10 mM KoAc, 1.5 mM Mg(OAc)2 , 2 mM dithiothreitol, 1 mM PMSF, and 200 U/ml RNaseOUT ribonuclease inhibitor [Thermo Fisher Scientific]), then ruptured by a Dounce homogenizer. .. To separate microsomal and cytosolic fractions, homogenates were subjected to low-speed centrifugation (1,500 ×g ) for 5 min.

    Ethanol Precipitation:

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: Reactions proceeded for 2 min, 5 min, or 15 min at room temperature (∼22°C) before being quenched by the addition of 3 µL of 1 M sodium acetate (pH 6), 1 µL glycogen, and 35 µL 95% ethanol, followed immediately by freezing on dry ice for 1 h and subsequent ethanol precipitation of the RNA. .. For reactions testing a dithiothreitol (DTT) quench, three separate quench solutions were prepared: DL-1,4 dithiothreitol (Acros Organics; 16568_0250) dissolved to 2.5 M in deionized water; 1 g of DTT dissolved in 5 mL of 1 M sodium acetate (pH 5); or 1 M sodium acetate (pH 5).

    Concentration Assay:

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: EDC stock solution (5.65 M; Sigma-Aldrich; 39391-10ML [listed as N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide]) was diluted to twice the desired final concentration in deionized water, and 5 µL of this diluted stock was added to the reaction mixture to give the desired final EDC concentration in a final reaction volume of 10 µL. .. For reactions testing a dithiothreitol (DTT) quench, three separate quench solutions were prepared: DL-1,4 dithiothreitol (Acros Organics; 16568_0250) dissolved to 2.5 M in deionized water; 1 g of DTT dissolved in 5 mL of 1 M sodium acetate (pH 5); or 1 M sodium acetate (pH 5).

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: In this study, nuclease-treated and purified FHV virions were supplemented with DMS to 5% final concentration. .. 100 U of TGIRT-III was mixed with 250 ng of RNA template, 0.5 mM of AzNTPs/dNTPs mixture (AzNTPs:dNTPs = 1:35), and the following reaction conditions: 5 mM dithiothreitol (Invitrogen), 10 U RNaseOUT (Invitrogen), 50 mM Tri–HCl pH 8.0, 75 mM KCl and 3 mM MgCl2 .

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: For reactions in a desired EDC concentration, 4–6 excised seedlings were placed in a 50 mL Falcon tube that contained buffer (HEPES, pH 7, HEPES, pH 8, or CHES, pH 9.2), KCl, and MgCl2 such that the addition of EDC diluted in deionized water gave a final total volume of 10 mL containing 50 mM pH buffer, 50 mM KCl, 0.5 mM MgCl2 , and EDC of the desired final concentration (110–565 mM). .. For treatments using a DTT quench, 1 g of DL-1,4 dithiothreitol (Acros Organics; 16568_0250) was added to the tube, which was then shaken vigorously for 2 min. Then, the reaction buffer was decanted and the seedlings were washed three times with ∼20 mL deionized water for each wash before immediate drying and quick freezing in liquid N2 .

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody
    Article Snippet: The guanidine HCl (24115) used for reduced mass analysis, trifluoroacetic acid (TFA, 28904), and dithiothreitol (DTT, 20290) were from Thermo Scientific. .. Optima LC/MS grade solvents, water (W6-4) and acetonitrile (ACN, A955-4), were from Fisher Scientific.

    Lysis:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: .. For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    Hood:

    Article Title: In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
    Article Snippet: All reactions involving EDC were performed in a chemical fume hood. .. For treatments using a DTT quench, 1 g of DL-1,4 dithiothreitol (Acros Organics; 16568_0250) was added to the tube, which was then shaken vigorously for 2 min. Then, the reaction buffer was decanted and the seedlings were washed three times with ∼20 mL deionized water for each wash before immediate drying and quick freezing in liquid N2 .

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  • 99
    Thermo Fisher dtt
    Dynamic range of <t>roGFP2-Orp1</t> and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM <t>DTT</t> at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p
    Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dtt - by Bioz Stars, 2020-03
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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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    Average 95 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    m dithiothreitol - by Bioz Stars, 2020-03
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    Image Search Results


    Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Journal: PLoS ONE

    Article Title: H2O2 dynamics in the malaria parasite Plasmodium falciparum

    doi: 10.1371/journal.pone.0174837

    Figure Lengend Snippet: Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Article Snippet: Purified recombinant roGFP2-Orp1 and HyPer-3/SypHer proteins were reduced with 5 mM DTT for 10 min and 20 mM DTT for 30 min, respectively, at 4°C, desalinated (ZebaTM Spin Desalting Columns, Thermo Scientific), and diluted in reaction buffer to a final concentration of 5 μ M. A 5-fold drug/redox-active compound dilution (25 μ l) was mixed with 100 μ l of 5 μ M roGFP2-Orp1/HyPer in a 96-well microplate (black, half-area, Greiner Bio-One, Frickenhausen).

    Techniques: Transfection, Lysis, Confocal Laser Scanning Microscopy, Fluorescence, Incubation

    Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p

    Journal: Scientific Reports

    Article Title: Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes

    doi: 10.1038/s41598-017-10093-8

    Figure Lengend Snippet: Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p

    Article Snippet: Purified recombinant roGFP2-Orp1 protein was reduced with 20 mM DTT for 30 min at 4 °C, desalinated (ZebaTM Spin Desalting Columns, Thermo Scientific), and diluted in reaction buffer to a final concentration of 5 µM.

    Techniques: Transfection, Blocking Assay, Fluorescence, Confocal Laser Scanning Microscopy

    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