As detoxifying enzymes, tau class GSTs have many substrates. We chose the typical substrates for GST detoxification, including 1-chloro-2,4-dinitrobenzene (CDNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-nitrobenzyl chloride (NBC), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrophenyl acetate (4-NPA) (Sigma-Aldrich Corp., St. Louis, MO, USA). For GOPX detoxification, we chose two substrates, cumene hydroperoxide (Cum-OOH) and ethacrynic acid (ECA) (Sigma-Aldrich), to test the effect of G- and H-site residues on substrate activity. The activities toward CDNB, NBC, DCNB, and ECA were measured as described by Habig et al. [33 (link)], the activity toward NBD-Cl was measured as described by Ricci et al. [34 (link)], and the activity toward Cum-OOH was measured as described by Edwards and Dixon [35 ]. The structures of substrates are shown in Table 1 . All reactions were carried out at 25 °C using 60 mM GSH (Sangon Biotech). Each reaction was repeated three times, and experimental results were based on the mean values of three independent experiments.
Cumooh
Manufactured by Merck Group
Sourced in Germany
CumOOH is a laboratory equipment product manufactured by Merck Group. It is a device designed for specialized applications in research and scientific investigations. The core function of CumOOH is to facilitate specific tasks and procedures within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
4 npa, by Merck Group (1 mentions)
Ethacrynic acid eca, by Merck Group (1 mentions)
Glutathione (gsh), by Sangon (1 mentions)
Sodium phosphate, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by Merck Group (1 mentions)
T buooh, by Merck Group (1 mentions)
Terrific broth, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
C11 bodipy, by Thermo Fisher Scientific (1 mentions)
Infinite f200, by Tecan (1 mentions)
4 protocols using cumooh
1
Assessing Glutathione S-Transferase Detoxification
2
Biochemical Reagents Acquisition
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Terrific broth, Isopropyl β-D-thiogalactoside, sodium phosphate, hydrogen peroxide, thioflavin-T, t-BuOOH and CumOOH were obtained from Sigma Chemical (St. Louis, MO, USA).
3
Evaluating SETD7 Function in Prostate Cancer Cells
4
Lipid Peroxidation Monitoring with C11-BODIPY
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C11-BODIPY (581/591) (Life Technologies, D-3861, Darmstadt, Germany) is a fluorescent dye that can be used as a lipid peroxidation sensor. This fatty acid analogue is lipophilic and incorporates into biomembranes. After being oxidized, the fluorescent properties of C11-BODIPY shift due to the change from the reduced (λex = 540 nm/λem = 595 nm) to the oxidized (λex = 480 nm/λem = 520 nm) state. The adjusted index of BODIPY oxidation was calculated as follows:
C2C12 cells were seeded in black-walled, clear-bottom 96-well cell culture plates. C2C12 cells were first differentiated as described previously. Prior to the addition of BODIPY, cells were incubated with medium (CON) or taurine (TAUR). After 24 h, the medium was removed, and the cells were rinsed and treated with 10 µM BODIPY in medium for 30 min. Then, the medium was removed, and the cells were exposed to either 2 µmol/L iron(II) sulfate (Fe2+, Merck, Germany), 80 µmol/L cumene hydroperoxide (CumOOH, Sigma Aldrich, Germany) or a mixture of 2 µmol/L Fe2+ and 80 µmol/L CumOOH in DPBS at 37 °C for 1 h. Afterwards, cells were washed with DBPS, and fluorescence of adherent cells was measured using a microplate reader (TECAN infinite F200, Grödig, Austria).
C2C12 cells were seeded in black-walled, clear-bottom 96-well cell culture plates. C2C12 cells were first differentiated as described previously. Prior to the addition of BODIPY, cells were incubated with medium (CON) or taurine (TAUR). After 24 h, the medium was removed, and the cells were rinsed and treated with 10 µM BODIPY in medium for 30 min. Then, the medium was removed, and the cells were exposed to either 2 µmol/L iron(II) sulfate (Fe2+, Merck, Germany), 80 µmol/L cumene hydroperoxide (CumOOH, Sigma Aldrich, Germany) or a mixture of 2 µmol/L Fe2+ and 80 µmol/L CumOOH in DPBS at 37 °C for 1 h. Afterwards, cells were washed with DBPS, and fluorescence of adherent cells was measured using a microplate reader (TECAN infinite F200, Grödig, Austria).
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