AmG was synthesized in our lab. GO, aniline (ACS reagent, ≥99.5%), N,N-dimethylformamide (DMF, 99.8%), PMMA (Mw = 996 000 g mol−1), ethylenediamine (EDA, ≥99%), ammonium persulfate (APS, ≥98.0%), and 5-sulfosalicylic acid dihydrate (SSA, ≥99%) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Deionized (DI) water was used in all experiments.
5 sulfosalicylic acid dihydrate
Manufactured by Merck Group
Sourced in Canada, United States
5-sulfosalicylic acid dihydrate is a chemical compound used in various laboratory applications. It is a crystalline solid that is soluble in water and other polar solvents. The compound has a chemical formula of C₇H₆O₅S·2H₂O and a molar mass of 254.22 g/mol.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium persulfate, by Merck Group (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Aniline, by Merck Group (2 mentions)
Ethylenediamine, by Merck Group (2 mentions)
Detectx glutathione fluorescent detection kit, by Arbor Assays (1 mentions)
N n dicyclohexylcarbodiimide, by Merck Group (1 mentions)
Chlorzoxazone, by Merck Group (1 mentions)
Cinnamoylglycine, by Merck Group (1 mentions)
Apap d4, by Merck Group (1 mentions)
2 benzoxazolinone, by Merck Group (1 mentions)
Acetaminophen, by Merck Group (1 mentions)
6 hydroxychlorzoxazone, by Cayman Chemical (1 mentions)
L phenylalanine d8, by Merck Group (1 mentions)
P cresol sulfate, by LGC (1 mentions)
T cinnamic acid, by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
Triton x, by Merck Group (1 mentions)
Phr1359, by Merck Group (1 mentions)
Bechmark, by Bio-Rad (1 mentions)
β nadph, by Merck Group (1 mentions)
5 protocols using 5 sulfosalicylic acid dihydrate
1
Synthesis and Characterization of AmG
2
Quantifying Glutathione Redox Status
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Plasma samples were mixed with an equal volume of cold 5% aqueous 5-sulfo-salicylic acid dihydrate (Sigma-Aldrich, St. Louis, MO, USA) solution to remove protein. The samples were incubated at 4°C for 10 min followed by centrifugation for 10 min at 14,000 rpm and 4°C. The supernatant was collected and stored at -80°C until analysis. GSH/GSSG concentration was measured using the DetectX® glutathione fluorescent detection kit (#K006, Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer’s instructions. This kit utilizes a nonfluorescent molecule, ThioStar, which covalently binds to the free thiol group on GSH to yield a highly fluorescent product. The fluorescent product is read at 510 nm in a fluorescence plate reader with excitation at 390 nm, and the results are expressed in μM. The samples were diluted 1:2.5 with assay buffer and 1:10 with sample diluent to produce a final dilution of 1:50 before assaying.
3
Bacterial Cellulose Nanopaper Synthesis
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Bacterial cellulose (BC) nanopaper was appropriated from Nanonovin Polymer Co. (Mazandaran, Iran). Amino-functionalized graphene (AmG) was synthesized in our lab. Graphene oxide (GO), aniline (ACS reagent, ≥99.5%), N,N-dimethylformamide (DMF, 99.8%), ethylenediamine (EDA, ≥99%), N,N′-Dicyclohexylcarbodiimide (DCC, 99%), ammonium persulfate (APS, ≥98.0%), and 5-Sulfosalicylic acid dihydrate (SSA, ≥99%) were all received from Sigma-Aldrich (Oakville, ON, Canada). Deionized (DI) water was used for all the experiments.
4
Acetaminophen Metabolite Analysis Protocol
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Acetaminophen, APAP-d4, Acetaminophen sulfate, cinnamoylglycine, acetate-13C6, propionate-13C6, chlorzoxazone, 2-benzoxazolinone, t-cinnamic acid, t-cinnamic acid-d6, l -phenylalanine-d8, 3-phenylpropionic acid (PPA), 3-phenylpropionic acid-d9, and 5-sulfosalicylic acid dihydrate were purchased from Sigma-Aldrich. Acetaminophen-d3 sulfate was from Santa Cruz Biotechnology. Acetaminophen glutathione disodium salt (APAP-GSH), 3-cysteinylAcetaminophen trifluoroacetic acid salt (APAP-CYS), butyrate-13C6, 3-phenylpropionic acid-13C3, p-cresol sulfate, and p-cresol sulfate-d7 potassium salt) were purchased from Toronto Research Chemical. 6-Hydroxychlorzoxazone was from Cayman Chemical. 3-(N-acetyl-l -cysteinyl) Acetaminophen disodium salt (APAP-mercapturate; APAP-NAC) was from Santa Cruz.
5
Quantifying Total Glutathione Levels
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The quantification of the total glutathione levels was performed in a 96-well plate, as described elsewhere [53 (link)]. The samples were homogenized in a solution containing an equal proportion of 0.1% Triton-X (catalog # 9002-93-1 or X100; Sigma-Aldrich, St. Louis, MO, USA) and 0.6% 5-sulfosalicylic acid dihydrate (catalog # 247006; Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 8000 × g for 10 min at 2–4°C to obtain the supernatant. The background wells, in triplicate, contained 20 μL of KPE (K2HPO4 buffer-EDTA, catalog # E9884; Sigma-Aldrich). The total glutathione was quantified in 20 μL of supernatants by adding 60 μL of the DNTB (5,5′-dithiobis (2-nitrobenzoic), catalog # D8113; Sigma-Aldrich, St. Louis, MO, USA) and 60 μL glutathione reductase (catalog # G3664; Sigma-Aldrich, St. Louis, MO, USA). After 30 s of incubation, 60 μL of β-NADPH (catalog # N1630; Sigma-Aldrich) was added to immediately read the 2-nitro-5-thiobenzoic acid formation at 412 nm every 30 s for 2 min using a microplate reader (Bechmark™, Bio-Rad, Hercules, California 94547, USA). The total glutathione concentration was determined via interpolation from a glutathione standard curve (26.4–0.4125 nM; catalog # PHR1359; Sigma-Aldrich, St. Louis, MO, USA). The results were expressed as μM/mg of total proteins [53 (link)].
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