Lipid peroxidation levels were measured by levels of malondialdehyde (MDA) using thiobarbituric acid method [27 (link)]. Kidney tissues were homogenized with 0.15M KCl (Duksan, Gyeonggi-do, South Korea) solution. 0.2ml of homogenous kidney tissue was added to 0.2ml of 8.1% SDS (Sigma-Aldrich, St. Louis, MO., USA) and incubated at room temperature for 10min. 3ml of 20% acetic acid (Duksan, Gyeonggi-do, South Korea)-0.8% TBA mixture (Lancaster Synthesis, Morecambe, England) (1:1, v/v) and 0.6ml of distilled water were added. The reaction mixture was heated in a water bath at 95℃ for 1h, and then cooled by tap water immediately. To each tube, 1ml distilled water and 5ml of n-butanol (Duksan, Gyeonggi-do, South Korea) and pyridine (Duksan, Gyeonggi-do, south Korea) (15:1, v/v) were added and shaken using a vortex. After centrifuging at 4,000 rpm for 10 min, the pink supernatant was measured at 532 nm using ELISA reader (BIO-TEK instruments, Winooski, VT, USA) with 1,1,3,3-tetramethoxypropane (Sigma-Aldrich) as a standard.
3 tetramethoxypropane
Manufactured by Merck Group
Sourced in United States, United Kingdom
3-tetramethoxypropane is a chemical compound used in various laboratory applications. It serves as a versatile building block for the synthesis of other organic compounds. The core function of this product is to provide a stable and reactive platform for chemical transformations and reactions. No further details on intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pyridine, by Duksan Pure Chemicals (1 mentions)
N butanol, by Duksan Pure Chemicals (1 mentions)
Elisa reader, by Agilent Technologies (1 mentions)
Potassium chloride (kcl), by Duksan Pure Chemicals (1 mentions)
2 thiobarbituric acid, by Merck Group (1 mentions)
Gemini em fluorescence microplate reader, by Molecular Devices (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
3 protocols using 3 tetramethoxypropane
1
Malondialdehyde Quantification in Kidney Tissue
2
Quantification of Lipid Peroxidation via TBARS
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As a marker of lipid peroxidation, 2-thiobarbituric acid-reactive substances (TBARS) were quantified [19 (link)]. Briefly, cells were treated in a lysis buffer (20 mM Tris-Cl, 2.5 mM EDTA, 1.0% SDS, pH 7.5). Cell lysates (200 mg protein in 100 μL) were mixed with 900 μL of 1.0% phosphoric acid and 1.0 mL of 0.9% 2-thiobarbituric acid (Sigma-Aldrich) and then heated on a boiling water bath for 45 min. Standard solutions of 1,1,3,3-tetramethoxypropane (Sigma-Aldrich), a precursor of malondialdehyde, were treated in the same way as cell lysates. After cooling, 1.5 mL of 1-butanol was added and the mixture was centrifuged at 13,000 rpm for 15 min to separate into two layers. The fluorescence intensity of the 1-butanol layer was measured at an emission wavelength of 590 nm (excitation at 540 nm) using the Gemini EM fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA).
3
Preparation of MDA-Modified LDL
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Preparation of MDA-LDL was carried out as described by Palinski et al [14 (link)]. Briefly, MDA was produced through rapid acid hydrolysis of 1,1,3,3-tetramethoxypropane (Sigma-Aldrich, Poole, UK) for 10 min at 37 °C using 4M of HCl. The pH was adjusted to pH7.4 using 1.5M NaOH creating a stock solution of 0.5M MDA. Human plasma LDL (Merck Millipore, Darmstadt, Germany) was incubated with MDA for three hours at 37 °C at a ratio of 100 μL of 0.5 MDA to 1 mg of LDL. ZebaSpin Desalting columns (7K MWCO; ThermoScientific, Loughborough, UK) were used to buffer exchange into PBS. Concentration of MDA-LDL was probed with the use of Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Loughborough, UK) according to manufacturer’s instructions. 0.01% EDTA was added to prevent further oxidation.
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