Cultures were centrifuged at 2879 g for 25 min at 4 °C, and the pellet was washed two times with 0.2 M ice cold sucrose. After the final wash, the cell pellet was disrupted by twice freeze–thawing and sonication, and resuspended in 1 mL TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea). Cell debris was removed by centrifugation at 19 940 g for 20 min at 4 °C. Membrane protein isolation was carried
out employing the ReadyPrep Protein Extraction kit (Membrane I) according to the manufacturer’s instructions (Bio-Rad Laboratories, Gladesville, NSW, Australia). Estimation of the MI-503 supplier protein content of the samples was performed using the bicinchoninic acid method employing a microtiter protocol (Pierce, Rockford, IL). Absorbances were measured using a Beckman Du 7500 spectrophotometer. Lysates (20 μg) were resuspended in SDS–PAGE sample buffer (0.375 M Tris pH 6.8, 0.01% SDS, 20% glycerol, 40 mg mL−1 SDS, 31 mg mL−1 DTT, 1 μg mL−1 bromophenol blue). For electrophoretic analyses, proteins were further denatured by heating at 100 °C for 5 min. Proteins were separated on 12% SDS–PAGE gels by electrophoresis for 2 h at 100 V. Gels were stained using Coomassie Brilliant Blue G-250 (Bio-Rad Laboratories) or transferred to methanol-treated
polyvinylidene difluoride membranes using the Trans-blot Selleckchem RXDX-106 cell transfer system (Bio-Rad Laboratories). Membranes were probed according to the Immun-Star™ WesternC™ kit
protocol (Bio-Rad Laboratories). Membranes were immunolabeled with patients’ sera, and goat anti-human IgG antibodies coupled to HRP (1 : 2000; Bio-Rad Laboratories) was used as a secondary antibody. Strip rehydration, isoelectric focusing, and SDS–PAGE were carried out according to the protocol supplied with the ReadyStrip IPG strips (Bio-Rad Laboratories). For each strip, protein aliquots (300 μg; 200 μg cytosolic those and 100 μg membrane extract) were suspended in 245 μL of a rehydration buffer consisting of 8 M urea, 100 mM DTT, 65 mM CHAPS, 40 mM Tris-HCl pH 8.0, 10 μL pH 4–7 and IPG buffer. Nuclease buffer (5 μL) was added, and the mixture was incubated at 4 °C for 20 min. The sample was then centrifuged at 7230 g for 15 min at 4 °C, and the supernatant was loaded for the first-dimension chromatography onto an 11-cm ReadyStrip IPG (Bio-Rad) of the appropriate pI range, and was left to incubate sealed for 24 h at room temperature. Isoelectric focusing was performed using an IsoeletrIQ™ Focusing System (Proteome Systems, Sydney, NSW, Australia). The machine was programmed to run at 300 V for 4 h, 10 000 V for 8 h, and 10 000 V for 22 h or until 80 000 Vh was reached.