Gdnhcl

CSA was performed as previously described [34 (link)] with slight modifications. Briefly, 10% brain homogenates from WTD or mice upon primary passage of the WTD isolates (3 different animals) were incubated with various concentrations (0 to 4 M) of GdnHCl (Sigma, Ca) for 1 hour at 20°C under shaking conditions (450 rpm). Then samples were treated with 50 μg/ml of PK for an additional hour at 37°C and the reaction was stopped by adding 1X pefabloc proteinase inhibitor. The samples were then subjected to Western blot and PrP^res signals were quantified as described above. The relative values of PrP^res (5 independent experiments) were plotted as a sigmoid curve against the GdnHCl concentration using GraphPad Prism (version 5). The GndHCL concentration required to denature 50% of PrP^Sc [GdnHCl_1/2] was deduced from these curves. The statistical analysis to compare the different isolates was performed using GraphPad Prism (version 5) software using unpaired student’s t-test.

Conformational stability assays were modified from a previous protocol (15 (link)). Aliquots of brain homogenates (15 μl) from each mouse of each group (n = 5 per group) were incubated for 1 h at room temperature with increasing concentrations of guanidine hydrochloride (GdnHCl; Sigma) ranging from 0 to 5 M in 0.5 M increments. Samples were precipitated in ice-cold methanol at −20°C overnight and centrifuged at 13,000 × g for 30 min at 4°C. Pellets were washed in PMCA buffer and centrifuged three times, resuspended in 18 μl of PMCA buffer, subjected to PK digestion, and Western blotted. Conformational stability was quantified by densitometric analyses of Western blots of triplicate samples from each mouse of each group, plotting the mean percentage of PrP^RES remaining ± standard deviation (SD) as a function of GdnHCl concentration and using fourth-order polynomial equations and nonlinear regression (GraphPad Prism) to fit denaturation curves for each prion strain.

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).