Silamat s5 shaker

Plant material, 100 mg of gametophytes at specific stages, was homogenized by adding glass beads to an Eppendorf tube and shaking with a Silamat S5 shaker (Ivoclar Vivadent, Schaan, Liechtenstein) twice during 10 and 5 s, respectively. Total RNA was isolated using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich, Buchs, Switzerland). DNA was removed using the TURBO DNA-free kit (Life Technologies, Carlsbad, CA), and checked to determine quality using the Bioanalyser Agilent RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany).

From each of the four samples (filamentous and heart tissues, two samples each) an amount of 20 mg dry weight of plant gametophytes were homogenized using a Silamat S5 shaker (Ivoclar Vivadent, Schaan, Liechtenstein). Homogenized samples were solubilized in 800 μL of buffer A [0.5 M Tris-HCL pH 8.0, 5 mM EDTA, 0.1 M Hepes-KOH, 4 mM DTT, 15 mM EGTA, 1 mM PMSF, 0.5% PVP and 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland)] using a Potter homogenizer (Thermo Fisher Scientific, Bremen, Germany). Proteins were extracted in two steps: first, the homogenate was subjected to centrifugation at 16,200 g for 10 min at 4°C on a tabletop centrifuge and, second, the supernatant was subjected to ultracentrifugation at 117–124 kPa (~100,000 g) for 45 min at 4°C. Post-ultracentrifugation the supernatant contained the soluble protein fraction. The pellet from the first ultracentrifugation was re-dissolved in 200 μL of buffer B (40 mM Tris base, 40 mM DTT, 4% SDS, 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland) to extract membrane proteins using the ultracentrifuge as described before. The supernatant after the second ultracentrifugation step contained the membrane protein fraction. Ultracentrifugation was performed using an Airfuge (Beckman Coulter, Pasadena, CA). Protein concentrations were determined using a Qubit Fluorometer (Invitrogen, Carlsbad, CA).