Gpx assay kit

A pathway-focused RT² Profiler PCR array (PAMM-069 customized to include Gsto2, Adh7, Sod3 and Gstm7) was used to analyze oxidative stress gene expression and first strand cDNA synthesis and real-time PCR conditions were performed in accordance with the manufacturer’s instructions (SABiosciences/Qiagen, MD). PERP, Actin and Alkbh8 gene expression were detected using Taqman Gene Expression assays and all qRT-PCR was carried out using an Applied Biosystems StepOne Plus detection system, with each sample tested in triplicate. Commercial antibodies used in this study were as follows: total p53 and GAPDH (Calbiochem, MA), Gpx1 and Alkbh8 (Santa Cruz Biotechnology, CA), Gpx3 and 6 (Abcam, MA) and Trx1 and 2 (Pierce, IL). Gpx activity was measured in whole cell lysates using a Gpx assay kit (SIGMA, MO), according to the manufacturer's specifications and using a total of 200 μg of protein in 250 μL for activity in units/mL.

Glutathione peroxidase activity was determined indirectly by the coupled reaction with glutathione reductase using a GPx assay kit (Sigma–Aldrich Chemie, Steinheim, Germany) in accordance with the methods previously described by Paglia and Valentine, Kaya et al. ( 18 (link) , 24 (link) ). Oxidized glutathione was converted to the reduced state by glutathione reductase, which was accompanied by the oxidation of NADPH to NADP with a decrease in absorbance of 340 nm. One unit of the enzyme that causes the oxidation of NADPH per min at 25°C is defined as an enzyme activity unit, as we previously described ( 18 (link) ).

Tissues were homogenized in 0.05 M phosphate buffer, pH7.0 and then centrifuged at 4000 ×g for 20 min at 4°C. The supernatants were used for GP_x and GR activity assay. The GP_x and GR activities were assayed with a commercial GP_x assay kit (Sigma, USA) and a GP assay kit (Sigma, USA), respectively. One unit of GP_x and GR activity was defined as the amount of sample required to oxidize 1 mmol of NADPH per minute based on the molecular absorbance of 6.22 × 10⁶ for NADPH.