To isolate the membrane fraction the T. gondii ghosts were resuspended in an equal volume of 2% thioglucopyranoside in 40 mM Tris pH 7.6 by pipeting the mixture up and down 10 times (suspension kept on ice) followed by a brief vortex.
Not all traits were recorded on all animals.> -wrap-foot> The dairy data used in this study were collected from the milk records kept on the farms and follow the trait descriptors commonly used by organisations such as National Milk Records Ltd (Harrogate, North Yorkshire, UK).
Non-actionable, reportable nodules were kept on regular (yearly) follow-up, indeterminate nodules were put on a more rapid follow-up of 3 6 months, while actionable nodules led to direct medical workup.
Non-actionable, reportable nodules were kept on regular (yearly) follow-up, indeterminate nodules were put on a more rapid follow-up of 3 6 months, while actionable nodules led to direct medical work-up.
The cell lysate was mixed with equal volume of phenol/chloroform and kept on ice for 5 min followed by spinning for 20 min at 10000 rpm and 4°C.
The sample was kept on ice for 30 minutes, followed by centrifugation at 14,000×g for 5 minutes, and the supernatant, identified as nuclear extract, was frozen at −80°C.
Acid extracts were prepared collecting cells in 5 10 volumes of Lysis Buffer H (10 mM Hepes pH 7.9, 150 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1.5 mM PMSF); Perchloric acid 0.2 M was added and cells were kept on ice for 30 minutes, followed by centrifugation for 10 minutes at 11000 g at 4°C; the supernatants were stocked at −80°C.
The resuspended pellet was then kept on ice for 15 min followed by centrifugation for 5 min.
The protein was then precipitated by adding 30 μL of 11.3% trichloroacetic acid and samples were kept on ice for 5 min followed by a short fast spin at 4 °C.
The reaction was stopped by adding 150 μl of 20% trichloroacetic acid (TCA) and kept on ice for 30 min, followed by centrifugation at 12,500 × g for 30 min. Then, the supernatant (250 μl) was mixed with equal volume of 2.5 M NaOH and the absorbance at 405 nm was measured.
Briefly, leaf samples were ground into powder in liquid nitrogen with a pestle and mortar and ~0.1 g of powder was homogenized in 1 ml 5 % pre-cooled perchloric acid (PCA) containing 0.5 g/L dithiothreitol (DTT), and kept on ice for 30 min, followed by centrifugation at 13,400 × g for 15 min at 4 °C.
