Phenylmethanesulphonyl fluoride

Rat liver tissue (20 g) was homogenised using rotor stator homogeniser in ice cold 1/3 strength phosphate buffered saline (PBS; 45.6 mM NaCl, 0.9 mM KCl, 2.7 mM Na₂HPO₄ and 0.48 mM KH₂PO₄ in distilled water, pH adjusted to 7.4 with HCl), in ratio 2:1 (PBS:tissue). Immediately before homogenisation, protease inhibitor phenylmethane-sulphonyl fluoride (Sigma) was added to a final concentration of 1 mM. Homogenate was then centrifuged using a bench top centrifuge for 5 min at 600g. The supernatant was collected and then re-centrifuged for 20 min at 3000g. The supernatant was collected again and was finally re-centrifuged using an ultracentrifuge at 100,000g for 4 h after which clear supernatant containing soluble proteins was collected.
Protein content was measured using bicinchoninic acid (BCA) assay. Protein content of samples was adjusted to 1 mg/ml prior to irradiation. Samples (10 ml aliquots) were irradiated at a distance of approximately 15 cm from the UV lamp (I=1.74×20 mW/cm², P=250 W, UV range 280–315 nm, IUV250 UV Curing Flood Lamp 230 V/50 Hz) for 0, 5 and 15 min respectively. After irradiation, protein damage was detected using carbonyl ELISA. Irradiated protein solutions (1 ml) were dried under vacuum centrifuge for 8 h with desferrioxamine added (5 mM) and stored at −80 °C.

Spot urine samples (15 mL) collected from human subjects, were first centrifuged at 10 000 r.p.m. for 10 min at 4°C in a RP-5B Refrigerated Superspeed Centrifuge to eliminate all debris and cells. Afterwards 16 mL of methanol (Sigma-Aldrich), 4 mL of chloroform (Sigma-Aldrich) and 12 mL of ddH₂O were added to 4 mL of cleared urine. The solution was further centrifuged at 6500 r.p.m. for 20 min at 10°C. Protein content in the lower phase was isolated and resuspended in 12 mL of methanol. After centrifugation at 65 000 r.p.m. for 50 min the supernatant was discarded and the pellet was completely air-dried and resuspended in 150 µL of lysis buffer containing 0.3 M sucrose (Sigma-Aldrich), 2 µM leupeptin (Sigma-Aldrich), 1 mM phenylmethanesulphonyl fluoride (Sigma-Aldrich), 25 mM imidazole (Sigma-Aldrich) and 1 mM EDTA (Sigma-Aldrich). Urine samples were diluted with the loading buffer and normalized to urinary creatinine. Immunoblotting was carried out as described above.