Mini gel electrophoresis unit

The fraction E, obtained with oleuropein (1%) as a sole carbon source, was added with (NH₄)₂ SO₄ 80% (w/v) to precipitate proteins. After overnight precipitation, the precipitates formed were collected by centrifugation at 10000 × g for 20 min at 4 °C, and the pellets obtained were dissolved in 20 mM sodium acetate buffer pH 5.0. The sodium dodecyl sulfate (SDS) gel was prepared with 0.1% SDS in 15% separating gels and 5.0% stacking gels (final gel concentration). Tris-glycine buffer pH 8.3 containing 0.1% SDS was used as the electrode buffer. Discontinuous SDS-PAGE in reducing conditions was performed according to the procedure of Laemmli [23 (link)]. Samples were treated with Laemmli buffer and boiled for 10 min at 95 °C before application to the gel. Electrophoresis was run from cathode to anode at 130 V, 30 mA for 40 min at room temperature in a Mini-Gel Electrophoresis Unit (Bio-Rad). The following proteins were used for calibration: lysozyme (14.6 kDa), esterase (28 kDa), β-glucosidase (60 kDa), bovine serum albumin (66 kDa), glucose oxidase (160 kDa). After running electrophoresis, proteins in the gel were visualized by staining with silver nitrate, according to the method of Wray et al. [24 (link)].

The protein profiles of the soluble phase and sediment recovered from sediment analysis (two and four weeks) were analyzed by SDS-PAGE. Aliquots of samples (10 μL) were loaded onto a precast polyacrylamide gradient gel (Any kD^TM, Bio-Rad Laboratories, Richmond, CA, USA) and separated in a Bio-Rad mini gel electrophoresis unit at 40 V. The protein bands were visualized using the Bio-Rad ChemiDoc XRS + system.