(A) SYPRO Ruby-stained 15% SDS-PAGE of purified GPI-anchorless PrP 27–30. The molecular mass of the purified peptide (residues 89–232) is 17,148 Da. The doublet seen at ~17 kDa reflects the presence of PK-resistant fragments of slightly different sizes (“ragged ends” []). Faint bands of ~34 kDa are characteristic dimers, the result of an incomplete dissociation of PrP 27–30 fibrils. Minor impurities correspond to traces of ferritin, tubulin and collagen [], which are clearly recognizable in electron micrographs. (B) Western blot of: Lanes 1–2, RML-infected WT mouse brain homogenate (BH), ± PK digestion. WT PrP presents as three characteristic bands of di-, mono-, and unglycosylated protein, respectively. Lanes 3–4, RML-infected GPI-anchorless PrP transgenic mouse BH, ± PK digestion; lane 5, purified GPI-anchorless PrP 27–30, used for cryo-EM studies; the 10–15 kDa band corresponds to a minor population of N-terminally truncated PK-resistant fragments described by Vázquez-Fernández et al. []; lanes 6–7, WT mouse BH inoculated with purified GPI-anchorless PrP 27–30, ± PK digestion. (C) Kaplan-Meier survival analysis of: WT mice infected with GPI-anchorless PrPSc BH (red), survival time 153 ± 10 days (standard deviation); WT mice infected with purified GPI-anchorless PrP 27–30 (green), survival time 203 ± 9 days (standard deviation); WT mice inoculated with PBS as negative control (blue) (n = 6, P < 0.05, Gehan-Breslow-Wilcoxon test).