Isoproterenol (1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride, molecular weight: 247.72) and TMQ (2-isopropyl-5-methyl-1, 4-benzoquinone; TMQ) (CAS number 490-91-5, molecular formula C10H12O2, molecular weight 164.20, and purity 99%, Figure 1 ) were procured from Sigma Aldrich (St. Louis, MO, USA). The chemicals and reagents including bovine serum albumin (BSA), 5-sulfosalicylic acid (SSA), naphthylene diamine dihydrochloride, sulphanilamide, phosphoric acid, HEPES, sucrose, 1,4-dithiothreitol (DTT), CHAPS, sodium chloride, protease inhibitors, phenyl methyl sulfonyl fluoride (PMSF), Tween-20, sodium nitrate, 3,3,5,5′-tetramethyl benzidine (TMB), and reduced form of glutathione (GSH) assay kit were purchased from Sigma Aldrich. The enzyme linked immunosorbent assay (ELISA) kit was obtained from R&D Systems, USA. The aspartate transaminase (AST), alanine transaminase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) kits were procured form Roche Diagnostics, USA.
5 sulfosalicylic acid
Manufactured by Merck Group
Sourced in United States
5-sulfosalicylic acid is a chemical compound used as an analytical reagent in laboratory settings. It is a white to off-white crystalline solid that is soluble in water and alcohol. The compound has a molecular formula of C7H6O5S and a molar mass of 202.18 g/mol.
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Lab products found in correlation
Nadph, by Merck Group (5 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (5 mentions)
Glutathione reductase, by Merck Group (5 mentions)
Trichloroacetic acid, by Merck Group (4 mentions)
Ammonium acetate, by Merck Group (3 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Glutathione assay kit, by Merck Group (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Phosphoric acid, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Optima lc ms grade methanol, by Thermo Fisher Scientific (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
2 vinylpyridine, by Merck Group (2 mentions)
Hdtma, by Oakwood Chemical (2 mentions)
Chrome azurol s, by Thermo Fisher Scientific (2 mentions)
Piperazine hexahydrate, by Merck Group (2 mentions)
Iron 3 chloride hexahydrate, by Merck Group (2 mentions)
Sodium phosphate, by Merck Group (2 mentions)
44 protocols using 5 sulfosalicylic acid
1
Cardiac Stress Biomarker Evaluation
2
Quantifying Cellular Glutathione Levels
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Cells were plated at a density of 2×106 in 150 mm dishes. At 16 h, BSO was applied; at 24 h, TAK1 inhibitor; and at 25 h, NAC. At 48 h, cells were washed in cold PBS and then scraped into 200 µL of 5% 5-sulfosalicylic acid (Sigma-Aldrich, St. Louis, MO) in water. Total GSH content was measured as described previously [11] (link). Glutathione disulfide (GSSG), the oxidized form of GSH, was measured by adding 20 µL of a 1:1 mixture of 2-vinylpyridine and ethanol per 100 µL of sample, incubating for 2 h, and assaying as previously described [12] (link). Glutathione determinations were normalized to the protein content using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA). Overall values were normalized to vehicle control.
3
Intracellular Glutathione Quantification
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Following treatment of 1 × 105 cells for 24 h, media was aspirated, cells were washed with PBS, and pelleted and mixed with 150 µL of 5% sulfosalicylic acid (Sigma). Cells were lysed via two cycles of freeze-thaw, cell debris was pelleted by centrifugation and the glutathione content in the resulting supernatant was measured using a glutathione detection kit (Sigma). Separate dishes that were treated in parallel were used to quantify protein content, which was then used for glutathione normalization. Alternately, intracellular glutathione content was also measured in a 96-well format using the GSH/GSSG-Glo Assay (Promega) following a 24 h treatment with normalization performed to viable cell count.
4
Measurement of Cellular Glutathione Levels
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Parental cells and MTDH KO cells were plated in 100-mm dishes and allowed to grow for 24 h. Cells were then washed with cold PBS and scraped into 300 µL 5% 5-sulfosalicylic acid (Sigma) in water and stored at −20 °C for a maximum of 72 h. Total GSH content was determined as described previously32 (link),33 (link). GSH disulfide (GSSG) was determined by adding 35 μL of a 1:1 mixture of 2-vinylpyridine and ethanol to 175 μL of sample and incubating for 2 h before assaying. The rates of the reaction were compared with similarly prepared GSH and GSSG standard curves. GSH determinations were normalized to the protein content of the insoluble pellet from the 5-sulfosalicylic acid dissolved in 2.5% SDS in 0.1 N bicarbonate using the BCA Protein Assay kit (Thermo Scientific).
5
Glutathione Biosynthesis and Assays
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Caffeic acid (CA), 3,4-dihydrocinnamic acid (HCA), 3,4-dimethoxycinnamic acid (DMCA), 1-chloro-2,4-dinitrobenzene (CDNB), reduced glutathione (GSH), oxidized glutathione (GSSG), gly–gly, l -γ-glutamyl-p-nitronilide, NADPH, ATP, cysteine, glutamine, serine, 2,3 naphthalene dicarboxaldehyde (NDA), 5-sulfosalicylic acid (SSA), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), enzymes and other common reagents were purchased from Sigma Chemical Co., St. Louis, MO. Primary and secondary antibodies were purchased from Abcam, Cambridge, MA. RNA isolation kits, cDNA kits and qRT-PCR reagents were purchased from Agentek, Israel.
6
Biocatalytic Nanocomposite Synthesis
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Ascorbic acid, Cu (CuSO4·5H2O), d ‐glucose, fluorescein isothiocyanate (FITC), GOx (from Aspergillus niger), GSH, 2,2′‐biquinoline‐4,4′‐dicarboxylic acid dipotassium salt trihydrate (BCA), 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), 3,3′,5,5′‐tetramethylbenzidine dihydrochloride hydrate (TMB), 5‐sulfosalicylic acid, and 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB) were acquired from Sigma–Aldrich (St. Louis, MO). 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO) and ICG were supplied by Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). CNC (lyophilized form) was purchased from Cellulose Lab (Fredericton, NB, Canada). LAP (Laponite XLG) was obtained from BYK Additives & Instruments (Wesel, Germany).
7
Stable Isotope Labeled Metabolite Analysis
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[13C3]-sodium propionate was purchased from Cambridge Isotopes (Tewksbury, MA). 5-sulfosalicylic acid (SSA), trichloroacetic acid, and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO). Optima LC-MS grade methanol (MeOH), acetonitrile (ACN), formic acid, and water were purchased from Fisher Scientific (Pittsburgh, PA).
8
Glutathione Quantification in Muscle
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Glutathione levels in the gastrocnemius homogenates were determined using the DetectX Glutathione Fluorescent Detection Kit (Kit # K006, Arbor Assays, Ann Arbor, MI, USA). Initially, samples were diluted with an equal volume of ice-cold 5 % 5-sulfo-salicylic acid (catalog # S2130, Sigma) and left on ice for 10 min. Samples were centrifuged for 10 min at 21,000 × g after which 30 µl of the resulting supernatant was carefully removed, added to a fresh centrifuge tube and diluted with Assay Buffer provided in the kit. Diluted samples and standards were added to the plate at 50 µl/well followed by 25 µl of ThioStar™ Diluent provided in the kit. The plate was then incubated for 15 min at room temperature after which fluorescence was read using the BioTek Synergy Model H1M Multimodal Plate Reader with excitation at 370 nm and emission at 510 nm. This represents the free (reduced) glutathione concentration. After addition of the kit-provided “Reaction Mixture” (25 µl/well) and a second 15 min room temperature incubation, the plate was again read using the conditions described above. This reading represents the total glutathione concentration. The oxidized (GSSG) glutathione levels were obtained by subtracting the reduced glutathione from the total glutathione results.
9
Quantifying Glutathione Levels in Kidneys
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Kidneys were homogenized in 5% 5-sulfosalicylic acid (Sigma Aldrich). The samples were then assayed for total glutathione (GSH) content by the method of Griffith [36] (link). The protein levels determined using the BCA Assay Kit (Thermo Scientific, Rockford, IL). Values were normalized to the protein content of whole homogenates. Percent of total glutathione as glutathione disulfide was calculated. For more details, see the supplemental section.
10
Comprehensive Biochemical Assay Protocols
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STZ, sodium citrate, citric acid, bovine serum albumin, 5-sulfosalicylic acid (SSA), naphthalene diamine dihydrochloride, sulphanilamide, phosphoric acid, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sucrose, 1,4-dithiothreitol (DTT), CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, sodium chloride, protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), tween 20, sodium nitrate, 3,3,5,5′-Tetramethylbenzidine (TMB), glutathione (GSH) assay kit, and all other required chemicals, if not specified, were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). All chemicals used in the present study were of analytical grade. Malondialdehyde (MDA) assay kit was purchased from Northwest Life Science Specialties (WA, USA). Cytokines duo set ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). EnzChek myeloperoxidase (MPO) activity assay kit was purchased from Life Technologies (NY, USA).
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