Recovery of sarkosyl-insoluble fraction from human brains

A sarkosyl-insoluble fraction of human tau was recovered as described [²²,³¹–³³] with minor modifications. Briefly, 350 μL of the 10% homogenates were mixed with the same volume of A68 buffer to a final concentration of 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 1 mM EGTA, 5 mM EDTA, and 10% sucrose, followed by clarification at 13,000×g for 20 min. The supernatant was set aside and the pellets were resuspended in the same buffer and again subjected to centrifugation for 20 min. The supernatant portions from each centrifugation were combined, and sarkosyl was added to a final concentration of 1%. The mixture was incubated for 1 hour at room temperature on a flat rotating shaker set at 700 rpm, and subsequently centrifuged at 100,000×g for 1 hour at 4°C. The resultant pellets were suspended in PBS at 1/5^th the starting volume of the 10% homogenates, and sonicated and stored at 4°C until use. This final sarkosyl-insoluble fraction of AD brains were used for the seeded polymerization of tau expressed in cultured cells (see below).