Protein carbonyl content assay kit

Preparation of BSA^Ald and binding of Gd³⁺ probe to protein was carried out according to modified procedures (48 (link)). To a solution of bovine serum albumin (BSA) (100 mg) dissolved in phosphate buffered saline (4 mL, pH 7.4, 0.25 mM) was added sodium ascorbate (20 mg), ferric chloride (120 μL, 10 mM) and 20 μL H₂O₂ (30% w). The reaction was stirred at 37 °C for 24 h, and sodium ascorbate (20 mg) was added repeatedly every 8 h. After 24 h, the protein was purified using PD-10 Sephadex G25 desalting columns (GE Healthcare), eluted with PBS. Protein concentration was assessed using the ‘Micro BCA Protein Assay Kit’ (Thermo Scientific, 23235). Protein carbonyl concentration was determined by ‘Protein Carbonyl Content Assay Kit’ (Sigma-Aldrich, MAK094-1KT). BSA^Ald had an aldehyde concentration of 4 aldehyde/protein. The protein solutions were kept at a concentration of 20 mg/mL for further use.

Oxidative stress levels were measured by dichlorodihydrofluorescein (DCF) imaging, the ratio of reduced glutathione (GSH)/glutathione disulfide (GSSG) and protein carbonyl content assay. For DCF imaging, cells were incubated with 25μM 5-and-6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) at 37°C for 30 minutes. Hoechst 33342 was added at a final concentration of 1.0 μM to the carboxy-H2DCFDA staining solution during the last 5 minutes of the incubation. The cells were then imaged by Leica TCS SP5 (Leica Microsystems, IL). For GSH/GSSG ratio, GSH/GSSG-Glo assay from promega (Madison, WI) was used according to manufacturer’s guide. Five thousand live cells were seeded in triplicates in white wall 96-well plates 8 hours before the treatment to allow the cells to attach, right after the treatment, cells were washed and lysed in the plate and GSH/GSSG ratio was measured by luminescence signal. Protein carbonyl content assay kit was purchased from Sigma Aldrich (St. Louis, MO), Carbonyl content was determined by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of stable dinitrophenyl (DNP) hydrazone adducts, which was then detected spectrophotometrically at 375 nm, proportional to the carbonyls present. The measurements were carried out in duplicates.