All solvents, unless specified, were of analytical grade. Keratin was extracted from Australian Merino wool, 19.5▒µm fineness, by the sulphitolysis reaction, slightly modifying the extraction methods described in previous works [21,22]. Briefly, a fibre sample, withdrawn from a combed sliver and cleaned by Soxhlet extraction with petroleum ether, was washed with distilled water and conditioned at 20▒°C, 65% R.H. for 24▒h. Five grams of cleaned and conditioned fibres was cut into snippets some millimetres long, put in 100▒mL of aqueous solution containing urea (8▒M), selleck inhibitor metabisulphite (0.5▒M) and sodium

dodecyl-sulphate (SDS), adjusted to pH 6.5 with NaOH 5▒N under strong

mechanical shaking for 2▒h at 65▒°C using the Linitest apparatus. The Linitest consists of a water reservoir containing a rotative axle with two stainless steel vessels radially connected. The wool, immersed in the extraction solution, is put in the two vessels with some steel bails and the axle rotates at a frequency of 40±2▒rpm. The water temperature is thermostatically controlled and maintained constant during the test. The mixture was filtered with 5▒µm pore-size filter, using a peristaltic pump and dialyzed against distilled water using a cellulose tube this website (6500▒Da molecular cut-off) for 3 days at room temperature, changing distilled water four times a day. The keratin aqueous solution

obtained after dialysis was freeze-dried in order to obtain a keratin powder. Keratin membranes containing CER3 and CER6 in different ratios (Table 1) were prepared as described below: CER3 and CER6 were dispersed in formic acid using an ultrasound bath at the frequency of 50▒Hz for 2▒h. Successively, the keratin powder was added to the ceramides dispersion in order to obtain a solution at the keratin concentration of 5% w/w with respect to formic acid. The dissolution of keratin powder was performed under shaking for 2▒h at room temperature. Once keratin was dissolved, the solution was subjected to ultrasonic treatment for 2▒h in order to remove air bubbles. The solutions were cast in a 5×5▒cm2 polyethylene 4��8C mould at 50▒°C until constant weight. The mould was filled with 5, 7, and 10▒mL of solution, in order to obtain membranes having a thickness of about 60, or 140, or 180▒µm. The molecular weights of keratin powder were determined by electrophoresis SDS-PAGE. Before electrophoretic analysis, the keratin powder was dissolved in a reductive buffer of dithiothreitol/urea at pH 8.6 under nitrogen atmosphere for 4▒h. SDS-PAGE was performed according to Laemmli’s method [23] using XcellLock Mini Cell (Invitrogen, USA), on 12% polyacrilamide gels.