Proteinase k

Fibrils in growth buffer were incubated with a 1:10 molar ratio of freshly dissolved Proteinase K (GoldBio) to rPrP^94-178 monomer for 24 hours at room temperature without agitation^{67 (link)}. After the incubation period, any remaining insoluble material was pelleted by centrifugation at 16,160 xg for 5-7 minutes and the supernatant removed. The pellet was resuspended in water (for negative stain imaging) or 2 % SDS (for cryo-EM).
For a measure of insoluble character in response to Proteinase K treatment, dissolution experiments were monitored by nephelometry (Extended Data Fig. 2). A slurry of 100 μL of rPrP^94-178 fibrils in growth buffer with the addition of 1:10 molar ratio of Proteinase K: rPrP^94-178 monomer (10 μM ProK: 100 μM rPrP^94-178 monomer), growth buffer with the same amount of Proteinase K, growth buffer only, or water were added to a sealed 96-well plate and loaded into a NEPHELOstar Plus (BMG Labtech). Readings in nephelometry units (NTUs) were recorded at 37 °C for 0.1 seconds every 10 minutes for 24 hours with a 2.5 mm beam at 12 % intensity. Before each reading, the plate was shaken at 300 rpm for 20 seconds.