PrP Proteinase-K Digestion of Brain Homogenates

For proteinase-K (PK) digestion of crude brain homogenates, we used protein/enzyme ratios as previous reports [16 (link)]. Briefly, 20 μg of brain homogenate was incubated with 20 μg/mL of PK (New England Biolabs) for 1 h at 37 ⁰C. The reaction was then stopped by addition of Pefabloc (Sigma), and samples were boiled with SDS-sample buffer and electrophoresed on 4–12% NuPAGE gels. For PK digestion of asymmetric flow field-flow fractionation (AF4) fractions, 10 μL of each fraction was incubated with 20 μg/mL of PK in the presence of 20 μg bovine serum albumin (BSA) as the sacrificial substrate to account for the low mass of PrP in eluted fractions, and to assure constant total protein mass in all samples. Reactions were then adjusted to the same volume for all samples and they were incubated with PK for 1 h at 37 ⁰C. The reaction was then stopped by addition of Pefabloc. Samples were then boiled with SDS-sample buffer and separated by SDS-PAGE.