D8130

Manufactured by Merck Group

Sourced in Germany, United States

The D8130 is a laboratory equipment product. It serves a core function within the research and scientific community. However, a detailed description of its capabilities and intended use would require additional research to maintain an unbiased and factual approach. As a result, a comprehensive description cannot be provided at this time.

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CAS No D8130 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, D8130),

14 protocols using d8130

Evaluation of GSH-Px Activity in SGC-7901 Cells

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GSH-Px activity in SGC-7901 cells was evaluated by a previously described method, through a coupled assay using H₂O₂ and dithiobis-nitrobenzoic acid (D8130; Sigma-Aldrich; Merck Millipore). Enzymatic activity (1 U) represented a decrease in GSH concentration of 1 mmol/l/min following subtraction of non-enzymatic mode. All measurements were performed in triplicate, and the results were normalized per mg protein.

Jiang T., Zhang H., Liu X., Song H., Yao R., Li J, & Zhao Y. (2016). Effect of oxaliplatin combined with polyenephosphatidylcholine on the proliferation of human gastric cancer SGC-7901 cells. Oncology Letters, 12(6), 4538-4546.

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Quantifying TrxR1 Enzymatic Activity

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Enzymatic activity of purified TrxR1 variants was assessed colorimetrically by monitoring NADPH (N7505; Sigma)-dependent reduction of Ellman's reagent (DTNB, D8130; Sigma). A plate reader (Synergy H1 Hybrid Multi-Mode Reader, 11-120-534; BioTek) autodispenser was used to add DTNB (in phosphate buffer) to each well to start the reactions, which included final concentrations of 5 mM DTNB, 300 μM NADPH, and 50 nM (Fig. 3A) or 100 nM (Fig. 7B) TrxR1 in a final volume of 100 μL per well. This reaction was monitored at 412 nm every minute for 50 min. Reactions were performed in triplicate. For all activity assays, error bars display one standard deviation based on at least triplicate experiments. A control lacking the TrxR1 enzyme was conducted in triplicate and has been subtracted from all enzyme assays.

Wright D.E., Altaany Z., Bi Y., Alperstein Z, & O'Donoghue P. (2018). Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity. Antioxidants & Redox Signaling, 29(4), 377-388.

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Quantifying Acetylcholinesterase Activity

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Acetylcholinesterase activity was determined as described previously (Savina et al. 2002 (link)). Briefly, 100 μl of each fraction collected from the iodixanol density gradient was diluted with DPBS to 180 μl and incubated with 20 μl mixture of 1.25 mM acetylthiocholine (Sigma-Aldrich; A5751) and 0.1 mM 5,5′-dithiobis(2-nitrobenzoic acid) (Sigma-Aldrich, D8130) for 30 min at 37 °C. The absorbance was measured at 412 nm with spectrophotometer Synergy 2 Multi-Mode Reader (BioTek Inc., Germany) and the activity presented as pmol/ul*min.

Dominkuš P.P., Ferdin J., Plemenitaš A., Peterlin B.M, & Lenassi M. (2017). Nef is secreted in exosomes from Nef.GFP-expressing and HIV-1-infected human astrocytes. Journal of neurovirology, 23(5), 713-724.

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Enzymatic Activity Assay of CysK in C. difficile

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Enzymatic activity of recombinantly produced CysK of C. difficile was measured photometrically as previously described with slight modifications (Tai et al., 1993 (link); Saavedra et al., 2004 (link)). Briefly, the assays were performed in a 96-well format in 100 μL final volume. As substrates O-Acetyl-L-serine hydrochloride (OAS; Sigma-Aldrich^{^®}, A6262) and 5-Thio-2-nitrobenzoic acid (TNB) were used. 20 mM OAS stock solution was freshly prepared in 100 mM HEPES (pH 7.0). Fresh TNB-stock solution was prepared by dissolving 10 mM 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich^{^®}, D8130) and 15 mM dithiothreitol in 100 mM HEPES (pH 7.0). Each reaction contained 2 mM OAS and 50 μM TNB. Following the addition of CysK at a final concentration of 7 μM, the reactions were monitored in a VarioSkan^TM (Thermo Fisher, United States) plate reader at 30°C by measuring the OD₄₁₂_nm every 4 min. If needed CysK was denatured by incubating aliquots for 30 min at 56°C.

Ünal C.M., Karagöz M.S., Berges M., Priebe C., Borrero de Acuña J.M., Wissing J., Jänsch L., Jahn D, & Steinert M. (2019). Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses. Frontiers in Pharmacology, 10, 340.

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Citrate Synthase Activity Assay

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The activity of citrate synthase was measured based on the method described by Srere [33 ]. Porcine citrate synthase is the first enzyme of the citric acid cycle and exists in almost all cell and tissue types. It converts Acetyl-coA and oxaloacetate into citrate and releases reduced coenzyme-A. The latter reacts with the Ellman’s reagent (DTNB) enabling the reaction to be followed at 412 nm where reacted DTNB absorbs. The reaction was performed with 6 nM citrate synthase (C3260, Sigma), 0.45 mM Acetyl-coA (A2056, Sigma), 0.5 mM oxaloacetate (O4126, Sigma), and 0.1 mM DTNB (D8130, Sigma) in 50 mM Tris-HCl buffer supplemented with 250mM NaCl, at pH 7.5, and followed for 3 min at 30°C.

Hristozova N., Tompa P, & Kovacs D. (2016). A Novel Method for Assessing the Chaperone Activity of Proteins. PLoS ONE, 11(8), e0161970.

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ELISA Measurement of Prostaglandins

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Measurement of PGE₂ and PGF_2α was performed using an ELISA technique in which acetylcholinesterase-linked PG tracers were used as described previously (Asselin et al., 1996 (link)). Rabbit anti-PGE₂ (kindly provided by Dr. T. G. Kennedy, University of Western Ontario, Ontario, ON, Canada) and sheep anti-PGF_2α (BioQuant, Ann Arbor, MI, USA) were used as selective antibodies. The inter- and intra-assay coefficients of variation (n = 12) were 16 and 10%, respectively. Briefly, 50 μl of collected culture medium was placed in a 96-well plate coated with goat anti-rabbit (PGE₂) or rabbit anti-sheep (PGF_2α) secondary antibody. A volume of 50 μl from each the tracer and the respective primary antibody was added to each sample after which the samples were incubated overnight at room temperature. After washing each well, 200 μl of Ellman's reagent (69 mM acetylthiocholine iodide; Sigma A5751, 5 g) and 54 mM 5,5'-dithiobis (2-nitrobenzoic acid; Sigma D8130, 5 g) dissolved in 10 mM phosphate buffer pH 7.4 was added. The plate was incubated on a shaker in the dark at room temperature, the reactions between the bound enzyme tracer and the Ellman's reagent yield a yellow color that can be measured with a photometric plate reader. A standard curve was used ranging from 39 to 5000 pg/ml PG.

Alzamil H.A., Pawade J., Fortier M.A, & Bernal A.L. (2014). Expression of the prostaglandin F synthase AKR1B1 and the prostaglandin transporter SLCO2A1 in human fetal membranes in relation to spontaneous term and preterm labor. Frontiers in Physiology, 5, 272.

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Crosslinking Analysis of FlgE Proteins

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WT and mutant FlgE_FL proteins were prepared in crosslinking buffer (XLB, 40 mM Tris pH 8.5, 160 mM NaCl, 1 M (NH₄)₂SO₄) at a final concentration and volume of 10 μM and 20 μL, respectively, and incubated at 4°C for 48 hours. Stock solution of NEM (99%, 139675, Beantown Chemical) and DTNB (D-8130, Sigma) were prepared to a final concentration of 20 mM in 100% ethanol. NEM and DTNB treated samples were prepared in a similar manner to non-treated samples except for the addition of 1.0 μL of 20 mM NEM/DTNB to yield a final concentration of 1 mM. Non-treated control samples were supplemented with 1.0 μL 100% ethanol. Samples were quenched with 6x SDS loading dye containing excess BME and electrophoresed on a 4–20% Tris-Gly SDS-PAGE gel. The relative crosslinking percentages were determined by comparing HMWC band intensities to that of the monomer band using the image analysis software FIJI.^{51 (link)}

Lynch M.J., Miller M., James M., Zhang S., Zhang K., Li C., Charon N.W, & Crane B.R. (2019). Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook. Nature chemical biology, 15(10), 959-965.

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Murine Colitis Induction and Analysis

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The following reagents were obtained: ketamine (Dopalen injectable^®, Paulínia, Brazil), xylazine (Anasedan injectable^®, Paulínia, Brazil), Tween 80 (Synth^®, Diadema, SP, Brazil), hexadecyltrimethylammonium bromide (HTAB) (Sigma Aldrich^® H5882, Steinheim, Germany), o-dianisidine dihydrochloride (Sigma Aldrich^® D3252, Steinheim, Germany), dextran sulphate sodium salt (DSS)—colitis grade (36,000–50,000 MW, cod. 02160110-CF, MP Biomedicals, LLC, Solon, OH, USA), hydrogen peroxide (Synth^®), horseradish peroxidase (Sigma Aldrich^® P8250, Steinheim, Germany), trichloroacetic acid (TCA) (Sigma Aldrich^® T4885, Steinheim, Germany), reduced L-glutathione (Sigma Aldrich^® G-4251, Steinheim, Germany), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma Aldrich^® N-7505, Steinheim, Germany), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma Aldrich^® D-8130, Steinheim, Germany), and buffer RIPA (Sigma Aldrich^® R0278, Steinheim, Germany). IL-1β mouse, IL-10 mouse, CXCL-MIP-2 mouse, and TNF-α mouse were obtained from R&D Systems^®, Inc. (Minneapolis, MN, USA).

da Silva G.G., Braga L.E., de Oliveira E.C., de Carvalho J.E., Lazarini J.G., Rosalen P.L., Dionísio A.P, & Ruiz A.L. (2023). Evaluation of a Standardized Extract Obtained from Cashew Apple (Anacardium occidentale L.) Bagasse in DSS-Induced Mouse Colitis. Foods, 12(17), 3318.

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Quantifying Cellular Glutathione Levels

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Cellular GSH was assessed by HPLC analysis with fluorescence detection after derivatization with monobromobimane (mBrB, Calbiochem). For GSSG analysis, aliquots of samples were reacted with N-ethylmaleimide to mask reduced thiols and then with dithiothreitol to reduce disulfide bridges, according to [31 (link)]. In some experiments colorimetric analysis of total thiol was carried out with the Ellman's assay; briefly, 50 μg of test sample proteins in triplicate were mixed with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid); Sigma-Aldrich; D8130) suspended in PBS, pH 7.5, to a final concentration of 200 μM. After incubation for 5 min at 37 °C, the absorbance was measured at 412 nm and the final concentration of thiols was calculated against a calibration curve of GSH.

Bartolini D., Piroddi M., Tidei C., Giovagnoli S., Pietrella D., Manevich Y., Tew K.D., Giustarini D., Rossi R., Townsend D.M., Santi C, & Galli F. (2014). Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation. Free radical biology & medicine, 78, 56-65.

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Glutathione Measurements in Cell Cultures

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Cells were cultured in the 6-well plate. When the culture reaches 70–80% confluency, cells were treated with L-BSO (20 μM) for 12 hours, or treated with LA (20 mM), hydrogen peroxide (100 μM), PEITC (10 μM), PL (10 μM), Dox (10 μg/ml), or ATO (10 μM) for 4 hours respectively. Cells were rinsed twice with 2 ml ice-cold Ca²⁺-/Mg²⁺-free PBS. Cells were collected by tripsinization and lysed in 0.2 ml of ice-cold extraction buffer (0.1% Triton-X and 0.6% sulfosalicylic acid in KPE) followed by 4 cycles of freezing and thawing (1 minute in liquid nitrogen and 2 min in water bath at 37°C). Cell lysate was centrifuged and supernatant was collected for GSH and GSSG measurement according to the method described by Rahman et al.53 (link). The GSH assay is based on the chemical conjugation of GSH with 5,5′-Dithiobis(2-nitrobenzoic acid)[DTNB] (Sigma, D-8130). Total glutathione was measured by firstly reducing oxidized glutathione using glutathione reductase (Sigma, G-3664) and β-NADPH (Sigma, N-7505) followed by conjugation with DTNB. To measure GSSG, GSH was firstly covalently reacted with 2-vinylpyridine (Aldrich, 132292), then GSSG was measured as described above. Pierce® BCA protein assay kit was used for protein determination. All samples were run in triplicates. GSH and GSSG levels were expressed in nmol/mg protein.

Zhu C., Hu W., Wu H, & Hu X. (2014). No evident dose-response relationship between cellular ROS level and its cytotoxicity – a paradoxical issue in ROS-based cancer therapy. Scientific Reports, 4, 5029.

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