The contents of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) were determined spectrophotometrically, with maximum optical absorption at 586 nm, by measuring the product that formed during the reaction using the selective reagent 1-methyl-2-phenylindole (Aldrich, CAS Number 3558-24-5) in accordance with Gérard-Monnier et al. [27 (link)]. To construct the calibration curve, 1,1,3,3-tetraethoxypropane was used [17 (link)].
1 methyl 2 phenylindole
Manufactured by Merck Group
Sourced in United States
1-methyl-2-phenylindole is a chemical compound used as a reagent in various laboratory applications. It serves as a detection agent for certain organic compounds. The core function of 1-methyl-2-phenylindole is to act as a colorimetric indicator, providing a specific color change in the presence of specific analytes.
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Lab products found in correlation
Tetramethoxypropane, by Merck Group (5 mentions)
Methanesulfonic acid, by Merck Group (4 mentions)
4 nitrophenol, by Merck Group (2 mentions)
Hydrogen peroxide, by Merck Group (2 mentions)
Diallyl disulfide, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
Goat anti rabbit and anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Il 1β antibody sc 1250, by Santa Cruz Biotechnology (1 mentions)
4 nitrophenyl n acetyl β d glucosaminide, by Merck Group (1 mentions)
Diallyl disulphide, by Merck Group (1 mentions)
Dinitrophenylhydrazine, by Merck Group (1 mentions)
B6916, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Merck Group (1 mentions)
Trimethoxypropane, by Merck Group (1 mentions)
Fecl3, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
14 protocols using 1 methyl 2 phenylindole
1
Spectrophotometric Analysis of MDA and 4-HNE
2
Spectrophotometric Determination of MDA and 4-HNE
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The contents of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) were determined spectrophotometrically, with maximum optical absorption at 586 nm, by measuring the product that formed during the reaction using the selective reagent 1-methyl-2-phenylindole (Aldrich, CAS Number 3558-24-5) in accordance with [41 (link)]. To construct the calibration curve, 1,1,3,3-tetraethoxypropane was used.
3
Anti-Inflammatory Biochemical Assays
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STZ, dichloromethane, diallyl disulfide, hydrogen peroxide, 4-nitrophenol, 4-nitrophenyl-N-acetyl-β-d -glucosaminide, tetramethoxypropane, 2,4-dinitrophenylhydrazine, methanesulfonic acid, 1-methyl-2-phenylindole were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of the highest purity available. IL-1β antibody: sc-1250; IL-6 antibody: sc-57315; TGF-β1 antibody: sc-130348 and Actin antibody: sc-8432 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-NFkβ p65 GTX102090 and Ikβ beta GTX82797 were from GeneTex (Irvine, CA, USA). Goat anti-rabbit and anti-mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, USA).
4
Lipid Oxidation Markers Quantification
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CDs were obtained after extraction with chloroform-methanol. The spectrophotometric assay was performed at 234 nm [15 (link)]. CDs were quantified using a molar extinction coefficient of 2.7 × 104 M−1 cm−1, and the results are reported as nmol of CD/mg dry weight. LHP levels were evaluated using the assay conditions described by El-Saadani et al. [16 (link)]. The calibration curves were obtained using 1 mM t-butylhydroperoxide as standard. The concentration of LHP was calculated using the molar absorptivity of I3 measured at 365 nm (є = 2.46 ± 0.25 × 104●M-1● cm−1), and the results are expressed as nmol LHP/mg dry weight. MDA levels were evaluated using 15 mM 1-methyl-2-phenylindole (Sigma-Aldrich, MO, USA) for detection at 586 nm. The values obtained are expressed as nmol of MDA/mg dry weight [17 (link)]. The dry weight was determined according to the conditions described by Bernal et al. [18 ].
5
Quantification of Lipid Peroxidation Markers
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The total concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) was quantified spectrophotometrically at a wavelength of 586 nm as formerly mentioned (Gérard-Monnier et al., 1998 (link)). In brief, liver homogenate samples were prepared as previously described (Shaker et al., 2016 (link)), followed by mixing 0.4 ml of sample with 0.65 ml of the reagent mixture [10.3 mM 1-methyl-2-phenylindole (Sigma-Aldrich, USA; Cat. no. 404888) dissolved in acetonitrile and diluted with 32 μM FeCl3 dissolved in methanol in ratio 3:1] and 0.15 ml of 15.4 M methanesulfonic acid (Merck, Germany; Cat. no. 8060220250). The sample tubes were subsequently kept at 45 °C for a period of 40 min, cooled on ice and centrifuged at 4000 g for 10 min. The collected supernatants were measured spectrophotometrically against sample blanks. A standard calibration curve of tetramethoxypropane (Sigma-Aldrich, USA; Cat. no. 108383) was conducted to estimate the concentration of total MDA + 4-HNE.
6
Biochemical Reagents Acquisition Protocol
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Angiotensin II (Ang II), dichloromethane, diallyl disulphide, hydrogen peroxide, 4-nitrophenol, tetramethoxypropane, dinitrophenylhydrazine, methanesulfonic acid, and 1-methyl-2-phenylindole were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of the highest purity available.
7
Plasma Malondialdehyde Quantification
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Aliquots of plasma were used to measure MDA at 586 nm, and the values obtained are expressed as pmol carbocyanine per mg dry weight (14). 1-Methyl-2-phenylindole (Sigma-Aldrich, MO) was used as a standard.
8
Lipoperoxidation Assays in Lipoproteins
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Lipoperoxidation products were measured in isolated LDL-c and HDL-c lipoproteins. CDs were obtained after extraction with chloroform: methanol (2:1). A spectrophotometric assay was performed at 234 nm [15 (link)]. CDs were quantified using a molar extinction coefficient of 2.7 × 104 M− 1 cm− 1, and the results are reported as nmol of CD/mg dry weight. LHP levels were evaluated using the assay conditions described by El-Saadani [16 (link)]. The calibration curve was obtained using t-butylhydroperoxide 1 mM as the standard. The concentration of LHP was calculated using the molar absorptivity of I3 measured at 365 nm (є = 2.46 ± 0.25 × 104 ● M− 1 ● cm− 1), and the results are expressed as nmol LHP/mg dry weight. MDA levels were evaluated using 1-methyl-2-phenylindole (Sigma-Aldrich, MO, USA) 15 mM for detection at 586 nm. The values obtained are expressed as nmol of MDA/mg dry weight [17 (link)]. The dry weight was determined according to the conditions described by Bernal [18 ].
9
Colorimetric Malondialdehyde Quantification
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A 650-μL aliquot of culture medium was placed in an Eppendorf tube and added with 150 μL of 1-methyl-2-phenylindole (10 mM) (Sigma) and allowed to react with MDA to produce stable chromophores. The tubes were vortexed and added with 30 μL of HCL (37 %), shaken again, and incubated in the dark at 45 °C for 60 min to drive the reaction to completion. After incubation, the tubes were centrifuged at 300 × g for 15 min. Finally, the absorbance of the upper organic layer was measured in a plate reader at 586 nm. A standard curve of 4, 3, 2, 1, 0.5, 0.25 and 0.125 mM tetramethoxypropane (TMOP) (Sigma) was built. The result was expressed as μg/mg protein by the Bradford standard method (Sigma, B6916).
10
Protein Quantification Assay Protocol
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Streptozotocin, bovine serum albumin, bicinchoninic acid, CuSO4, trimethoxypropane, methanesulfonic acid, HCl, FeCl3, 1-methyl-2-phenyl indole, acetonitrile, methanol, acetone, dichloromethane were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). All other reagents were obtained from commercial sources.
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