Prion Protein Amplification by PMCA

Protein misfolding cyclic amplification (PMCA) was performed as previously described (71 (link)). Samples (n ≥ 3) in PMCA conversion buffer were placed into polypropylene tubes in a Misonix 3000 sonicator (Misonix, Farmingdale, NY). The average output of the sonicator was 165 W during each sonication cycle. A PMCA round consisted of 144 cycles of a 5-s sonication, followed by an incubation of 9 min 55 s at 37°C. After each round of PMCA, an aliquot of sonicated sample was added to fresh 10% (wt/vol) uninfected brain homogenate in PMCA conversion buffer before the next round of sonication. The ratio of seed to uninfected brain homogenate was 1:20 for the first round of PMCA, 1:10 for the second round, and 1:2 for the remaining rounds. Aliquots (n ≥ 3) of uninfected brain homogenate were included in all rounds of PMCA as a negative control.