Dithiothreitol (dtt)

Manufactured by Avantor

Sourced in United States, Belgium

DTT is a reducing agent commonly used in biochemistry and molecular biology applications. It functions by breaking disulfide bonds in proteins, which can help maintain the protein's native structure and activity.

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Lab products found in correlation

Urea, by Avantor (7 mentions) Sodium chloride (nacl), by Merck Group (5 mentions) Sequencing grade modified trypsin, by Promega (5 mentions) Sodium dodecyl sulfate (sds), by Avantor (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (3 mentions) Triton x 100, by Merck Group (3 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions) Hydrochloric acid (hcl), by Merck Group (3 mentions) Acetonitrile, by Merck Group (3 mentions) Hepes, by Merck Group (3 mentions) Protease inhibitor cocktail, by Avantor (2 mentions) Tween 20, by Merck Group (2 mentions) Urea, by Merck Group (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Sodium chloride (nacl), by Avantor (2 mentions) Acetonitrile, by Avantor (2 mentions) Sodium hydroxide (naoh), by Merck Group (2 mentions) Acetic acid, by Avantor (2 mentions) Ecl chemiluminescence, by Thermo Fisher Scientific (2 mentions)

55 protocols using dithiothreitol (dtt)

Cationic Peptide-DNA Stability Assay

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Dextran-sulfate (DS; Sigma, St. Louis, MO, USA) was added to the prepared complexes at three-fold charge excess relative to the peptide. At 0 min and after 24 h of incubation EtBr fluorescence was measured on Wallac 1420D scanning multilabel counter and dye displacement was calculated.
For study of DTT destabilization the peptide/DNA complexes were prepared with addition of 1× SYBR-Green followed by incubation of complexes with 200 mM DTT (Amresco, Ohio, OH, USA) for 1 h at 37 °C. The fluorescence was measured, and SYBR-Green displacement was calculated as described above.

Egorova A., Shtykalova S., Selutin A., Shved N., Maretina M., Selkov S., Baranov V, & Kiselev A. (2021). Development of iRGD-Modified Peptide Carriers for Suicide Gene Therapy of Uterine Leiomyoma. Pharmaceutics, 13(2), 202.

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Purification and Interaction of UHRF1 and LSH

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To express UHRF1 in bacteria, PCR products encoding UHRF1 were ligated into pGEX4T-1 vector. GST-UHRF1 fusion protein was expressed and purified from E. coli. To purify FLAG-tagged LSH protein from mammalian cells, the 293T cells were transfected with plasmid encoding FLAG-LSH for 48 h. The cells were collected and lysed in high salt Lysis buffer (25 mM Tris–HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1× protease inhibitor cocktail, 1 mM DTT). FLAG-LSH protein was then captured with anti-FLAG M2-affinity beads and eluted with FLAG-peptide elution buffer (100 μg/ml FLAG-peptides, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM EDTA, 1× protease inhibitor cocktail, 1 mM DTT). For pulldown assay to test in vitro binding of UHRF1 and LSH, 1 μg of purified FLAG-LSH and 2μg of bead-bound GST-UHRF1 beads were incubated in pulldown binding buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0. 1% Triton X-100, 1 mM EDTA, 8% glycerol, 1× protease inhibitor cocktail (MCE), and 1 mM DTT (Amresco)) for 6 h at 4°C. The resulting beads were washed three times with ice-cold pulldown wash buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0. 1% Triton X-100, 1 mM EDTA, 1× protease inhibitor cocktail (MCE) and 1 mM DTT (Amresco)). After extensive washing, complexes were boiled in 1 × SDS loading buffer and analyzed by western blotting and Coomassie blue staining.

Han M., Li J., Cao Y., Huang Y., Li W., Zhu H., Zhao Q., Han J.D., Wu Q., Li J., Feng J, & Wong J. (2020). A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association. Nucleic Acids Research, 48(21), 12116-12134.

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Protein Extraction and Quantification in Batters and Foams

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Protein compositions of freeze-dried batters and foams were determined by extracting them with (i) 0.05 M sodium phosphate buffer (pH 6.8) containing 2.0% (w/v) sodium dodecyl sulfate (SDS) (i.e. SDS medium) and
(ii) the same buffer also containing 2.0% (w/v) dithiothreitol (DTT, VWR International, Leuven, Belgium) (i.e.
SDS+DTT medium) and then separating them with size-exclusion (SE)-HPLC (Pycarelle et al., 2020) (link). SDS disrupts non-covalent interactions (Turro, Lei, Ananthapadmanabhan & Aronson, 1995) (link), while DTT breaks up disulfide bonds. The latter leads to solubilization of cross-linked protein molecules (Konigsberg, 1972) (link).
Proteins were extracted in duplicate from two independent freeze-dried batter or foam samples (see sections 2.2.3 and 2.2.5). Samples contained 1.0 mg protein/mL extraction solvent. Extracts were loaded onto an HPLC system. Details on the latter and on HPLC analyses can be found in Pycarelle et al. (2020) (link).
With the data obtained, the percentage of protein extractable in SDS medium (SDS EP) was calculated (Equation 1). Total areas were integrated from 0 to 11 min to avoid interference of the elution of DTT.
SDS EP (%) = x 100 total area of sample SDS extract total area of sample SDS + DTT extract

Pycarelle S.C., Bosmans G.M., Pareyt B., Brijs K, & Delcour J.A. (2021). Free wheat flour lipids decrease air-liquid interface stability in sponge cake batter. Food research international (Ottawa, Ont.), 140.

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Recombinant Protein Expression and Purification

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Sodium chloride, glycerol, sodium phosphate saline (PBS), isopropyl β-d-1-thiogalactopyranoside (IPTG), Tris HCL and base, Glycine, lysozyme, dithiothreitol (DTT), and ethylene-di-aminetetraacetic acid (EDTA), Coomassie G-250 were purchased from VWR life science. Inclusion body (IB) solubilizing and protein refolding reagents: GSH, GSSG, L-Arginine, and urea were from Sigma-Aldrich (St. Louis, MI, USA)). PCR cloning kit: PET series vectors; all restriction enzymes; T4 DNA ligase and rapid protein assay BCA kit were from New England Biolabs (NEB, Ipswich, MA, USA) and Thermo Scientific (Cincinnati, OH, USA). ELISA reagents: Human serum albumin (HAS), bovine serum albumin (BSA), Tween-20, Citric acid, H₂O₂, o-phenylenediamine dihydrochloride (OPD) were from Sigma-Aldrich and Alfa Aesar. Protein purification apparatus and affinity chromatography columns were purchased from GE Healthcare.

Ban B., Sharma M, & Shetty J. (2020). Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts. Antibodies, 9(3), 39.

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Tissue Homogenization and DNA Extraction

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Tissues (15-35mg) were homogenised in 600μl of ATL lysis buffer (Qiagen Hilden, Germany) supplemented with 40mM—dithiothreitol (VWR Pennsylvania, United States), using a Tissue Ruptor (Qiagen). The homogenised solution was centrifuged at 14 000g for 3 mins to remove any micro-tissue particulate from the solution. From the recovered lysate 180μl was taken for DNA purification.

Nweke E.E., Naicker P., Aron S., Stoychev S., Devar J., Tabb D.L., Omoshoro-Jones J., Smith M, & Candy G. (2020). SWATH-MS based proteomic profiling of pancreatic ductal adenocarcinoma tumours reveals the interplay between the extracellular matrix and related intracellular pathways. PLoS ONE, 15(10), e0240453.

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Biocatalytic Alcohol Dehydrogenase Assay

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MES hydrate, triton X-100 (laboratory grade), acetaldehyde (ACS reagent, ≥99.5%), β-nicotinamide adenine dinucleotide [reduced disodium salt hydrate ≥97% (HPLC)], thiamine pyrophosphate, alcohol dehydrogenase (Deinococcus radiodurans recombinant from E. coli ≥10,000 units mL⁻¹), sodium pyruvate and polyvinylpyrrolidone were purchased from Sigma-Aldrich (Overijse, Belgium). Dithiothreitol was obtained from VWR International (Leuven, Belgium) and magnesium chloride from Chem-Lab Analytical (Zedelgem, Belgium).

Boeckx J., Hertog M., Geeraerd A, & Nicolai B. (2017). Kinetic modelling: an integrated approach to analyze enzyme activity assays. Plant Methods, 13, 69.

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Breast Cancer MCF-7 Cell Culture

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Human breast cancer MCF-7 cells were obtained from the ATCC and cultured in DMEM basal medium at 37 °C in 5% CO₂. The medium was supplemented with foetal bovine serum (10%), amphotericin B (2.5 μg/mL), and Penicillin-Streptomycin (50 U/mL). Chlorpromazine hydrochloride, fluphenazine dihydrochloride, thioridazine hydrochloride, anisomycin, H₂O₂, DMSO, HEPES buffer pH 7.5, NaCl, Triton X-100, bromophenol, MgCl₂, EDTA, Protease Inhibitor Cocktail, and PhosSTOP™ were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Gibco™ BenchStable™ DMEM, Gibco™ Fetal Bovine Serum (qualified, heat-inactivated, United States), amphotericin B (250 µg/mL), Penicillin-Streptomycin (5000 U/mL), and StemPro^® Accutase^® Cell Dissociation Reagent were obtained from Thermo Fischer Scientific (Waltham, MA, USA). Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) Electran^® for electrophoresis and dithiothreitol were obtained from VWR (Randor, PA, USA). Glycine was obtained from Alfa Aesar (Haver Hill, Fullerton, CA, USA), while sodium dodecyl sulfate was obtained from Merck (Darmstadt, Germany). INR119 was synthesized as previously described [14 (link)].

Otręba M., Sjölander J.J., Grøtli M, & Sunnerhagen P. (2021). A Small Molecule Targeting Human MEK1/2 Enhances ERK and p38 Phosphorylation under Oxidative Stress or with Phenothiazines. Life, 11(4), 297.

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Bovine Raw Milk Proteome Analysis

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Bovine raw milk was obtained from a local organic farmer. Microbial transglutaminase ACTIVA MP (EC 2.3.2.13) was from Ajinomoto (Hamburg, Germany). Sodium azide was purchased from Acros (Geel, Belgium). Tris(hydroxymethyl) aminomethane was obtained from AppliChem (Darmstadt, Germany), tri-potassium citrate monohydrate from GPR Rectapure (Leuven, Belgium) and potassium chloride from Carl Roth (Karlsruhe, Germany). Formic acid, sodium hydrogen phosphate dehydrate, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydroxide and sodium acetate were all purchased from Grüssing (Filsum, Germany). Hydrochloric acid, monopotassium phosphate, tri-sodium citrate dehydrate and potassium sulfate were obtained from Merck (Darmstadt, Germany). 3-[(3-Cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS) was bought from Molekula (Dorset, UK). Trypsin from bovine pancreas for sequencing was obtained from Sigma-Aldrich (Steinheim, Germany). Dithiothreitol (DTT), acetonitrile, urea, sodium chloride, acetic acid glacial as well as ethanol were all purchased from VWR (Darmstadt, Germany). Double-distilled water, prepared by Bi-Distillation

Duerasch A., Konieczny M, & Henle T. (2022). Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase. European food research and technology = Zeitschrift fur Lebensmittel-Untersuchung und -Forschung. A, 248(10).

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Osteogenic Cell Culture Protocols

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Gibco αMEM nucleosides glutaMAX, Gibco DMEM
hi-glucose glutamax media, bovine serum albumin (BSA), AlamarBlue,
Gibco 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
Gibco nonessential amino acids (NEAA), penicillin-streptomycin (10.000
U mL^–1), Gibco fetal bovine serum (FBS) and goat-antimouse
(GaM) secondary antibody (Alexa Fluor 488) were supplied by Fisher
Scientific. Glycine, gelatin (porcine skin, type A, gel strength 300),
allyl glycidyl ether (≥99%), Β-glycerophosphate, proline, l-ascorbic acid-2-phosphate sesquimagnesium salt (AsAp), dexamethasone,
acetic acid (≥99.8%), hydrochloric acid (HCl) (37%), sodium
chloride (NaCl) (≥99%), sodium hydroxide (NaOH) (>98%),
and
lithium phenyl-2,4,6-trimethylbenzoylphosphinate were obtained from
Sigma-Aldrich (Merk). Dialysis tubing cellulose membrane (MWCO 1000
Da), dithiothreitol, and molecular probes 6-diamidino-2-phenylindole
(DAPI) and rhodamine-phalloidin for F-actin were sourced from VWR.
Primary antibodies for anti-osteopontin (mouse) and anti-collagen
type I antibody (rabbit) were purchased from Developmental Studies
Hybridoma Bank. Donkey-antirabbit (DaR) secondary antibody (Alexa
Fluor 594) was purchased from Abcam.

Reizabal A., Saiz P.G., Luposchainsky S., Liashenko I., Chasko D., Lanceros-Méndez S., Lindberg G, & Dalton P.D. (2023). Cryo-Electrohydrodynamic Jetting of Aqueous Silk Fibroin Solutions. ACS Biomaterials Science & Engineering, 10(3), 1843-1855.

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Wheat Flour and Gluten Characterization

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Crousti wheat flour [12.0% protein; 14.0% moisture] was from Dossche Mills (Deinze, Belgium) and commercial gluten [Amygluten; 77.0% protein (N x 5.7); 6.0% moisture] from Syral (Aalst, Belgium). Salt and sugar were from a local supermarket. Fresh yeast was from AB Mauri (Merelbeke, Belgium). All chemicals were at least of analytical grade. Acetic acid, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), urea, disodium hydrogen phosphate and sodium dihydrogen phosphate were from VWR International (Leuven, Belgium). Bovine milk α-lactalbumin was from Acros Organics (Geel, Belgium). All other chemicals, reagents and molecular mass markers were from Sigma-Aldrich (Bornem, Belgium). Aq1 was from Puratos (Groot-Bijgaarden, Belgium).

Verbauwhede A.E., Lambrecht M.A., Fierens E., Hermans S., Shegay O., Brijs K, & Delcour J.A. (2018). Thermo-reversible inhibition makes aqualysin 1 from Thermus aquaticus a potent tool for studying the contribution of the wheat gluten network to the crumb texture of fresh bread. Food chemistry, 264.

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