Identification of sarkosyl-insoluble proteins that accumulate in the brains of aged mice.

Five young (2-month-old) and five aged (24-month-old) mouse brains were bisected along the midline to separate the right and left hemispheres, then snap-frozen in liquid nitrogen. The frozen tissues were pulverized using a Multi-Beads Shocker (Yasui Kikai) and stored at -150°C until further use.

The brain powder was homogenized in sarkosyl buffer (1% sarkosyl, 10 µg/mL RNase, 1 mM PMSF, 1 mM EDTA in PBS) using a Potter-Elvehjem homogenizer at 800 rpm. After a 10-minute incubation on ice, the homogenate was centrifuged at 15,000 g for 5 minutes at 4°C to separate the supernatant and pellet (P1). The supernatant was centrifuged again at 15,000 g for 5 minutes at 4°C, followed by a third centrifugation at 15,000 g for 15 minutes at 4°C to obtain the final supernatant (S1) and a pellet. S1 was then subjected to ultracentrifugation at 100,000 g for 1 hour at 4°C, resulting in a supernatant (S2) and a pellet. This pellet was resuspended in sarkosyl buffer using a vortex mixer and centrifuged again at 100,000 g for 1 hour at 4°C. The supernatant was discarded, and the final pellet (P2), representing the sarkosyl-insoluble fraction, was retained.