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. 2010 Sep 14;5(9):e12723. doi: 10.1371/journal.pone.0012723

Figure 3. Conformational stability and solubility assay in healthy and diseased voles.

Figure 3

A and B: Western blot analysis of scrapie brain homogenate (SBH) after denaturation with various concentrations of GdnHCl and separation of insoluble (A) and soluble (B) fractions by centrifugation. A: Immunoblot of pellets (P) at different concentrations of GdnHCl. In order to compare the fractions in the same blot, supernatant (S) at 0 M GdnHCl was loaded too. In the pellets, insoluble PrP decreased with increasing concentrations of GdnHCl (M). In each lane 0.4 mg TE were loaded. B: Immunoblot of supernatants at different concentrations of GdnHCl. In the first lane pellet (P) at 0 M GdnHCl was loaded too. In the supernatant PrP increased with increasing GdnHCl. In each lane 0.4 mg TE were loaded. Note that the FL and C1 PrP fragments are present in the supernatant after treatment with 0 M and 1 M GdnHCl, while the PrPSc-specific fragment C2 is visible in the supernatant from 1.5 M GdnHCl onwards, in parallel with the decrease of insoluble PrP in panel A. C: The conformational stability of PrPSc in SBH was analysed by denaturation curves best-fitted by plotting the fraction of PrPSc in the pellet (P) and in the supernatant (S), depicted in panel A and B respectively, as a function of GdnHCl concentration. [GdnHCl]1/2 values were 1.92 M in the pellet and 1.91 M in the supernatant fraction. D: Western blot analysis of supernatants (S) at different concentrations of GdnHCl from normal brain homogenate (NBH). In the first lane pellet (P) at 0 M GdnHCl was loaded. In NBH, PrPC was mostly in supernatant fraction and remained soluble at all GdnHCl concentrations tested. Samples were loaded as 0.4 mg tissue equivalent into each lane. A, B, D: Membranes were probed with SAF84. Molecular size markers are shown in kilodaltons on the left of each panel.