Conformational Stability of PrPSc

The conformational stability of PrP^Sc in brain homogenates from bank voles was analysed by CSSA as previously described [27 (link)]. Briefly, aliquots of brain homogenates (6% w/v in 100 mM TrisHCl, pH 7.4) were added with an equal volume of 100 mM TrisHCl (pH 7.4), sarcosyl 4% and incubated for 1 h at 37°C with gentle shaking. Then, aliquots of each sample were incubated for 1 h at 37°C with different concentrations of GdnHCl (Pierce) to obtain final concentrations ranging from 0 to 4 M. After GdnHCl treatment samples were centrifuged at 20,000 g for 1 h at 22°C and the pellets were re-suspended in denaturing sample buffer (NuPage LDS Sample Buffer and NuPage Sample Reducing Agent, Invitrogen) and analysed by western blot. The dose-response curves allowed us to estimate the concentration of GdnHCl able to solubilize 50% of PrP^Sc (GdnHCl_1/2). Individual denaturation curves were analyzed and best-fitted by plotting the fraction of PrP^Sc remaining in the pellet as a function of GdnHCl concentration, and using a four parameter logistic equation (GraphPad Prism).