TCA Precipitation for Protein Extraction

For TCA (trichloroacetic acid) precipitation, 10 mL of S. pombe cell culture (containing ∼1 × 10⁸ cells) was mixed with 1/4 volume (2.5 mL) of ice-cold 100% TCA. The resulting mixture was centrifuged, and pellets were washed with 10% TCA, followed by cell disruption with glass beads in 10% TCA. After centrifugation at 8000 rpm for 10 min at 4°, washed precipitates were resuspended in SDS sample buffer containing 1 mM phenylmethyl-sulfonyl fluoride (PMSF) and boiled at 70° for 10 min. After centrifugation at 14,000 rpm for 10 min, supernatants were loaded for SDS-PAGE. Antibodies against FLAG (Sigma), Mis17 (Shiroiwa et al. 2011 (link)), tubulin (TAT1; a gift from Dr. Keith Gull, University of Oxford, UK) and Cdc2 (PSTAIR; a gift from Dr. Yoshitaka Nagahama, National Institute for Basic Biology, Okazaki, Japan) were employed as primary antibodies. Custom-made 3–8% gradient Tris-Acetate gels (NuPAGE, Invitrogen) were used for Mis17, while NuPAGE 12% Bis-Tris gels were run using NuPAGE MES SDS Running Buffer for Cnp1. An LAS3000 (Fuji Film) was used for signal detection.