FIGURE 8.

Charge neutralization of the CLC allows more rapid generation of spontaneously arising (prion independent) amyloid fibrils by RT-QuIC. A, in unseeded (i.e. treated with normal brain homogenate (NBH)) RT-QuIC reactions, K₄A and K₄N rPrP 23–231 substrates had shortened lag times to spontaneous fibril formation compared with WT rPrP. The data points represent average percentages of maximum ThT fluorescence from four replicate wells as a function of reaction time. Similar rank orders of relative RT-QuIC kinetics were obtained in six experiments. B, Western blot analysis of unseeded (NBH treated), PK-treated RT-QuIC products using antisera to residues 90–104 or 218–231 (R30 and R20, respectively). Extended 17-kDa PK-resistant core fragments reactive with both antisera were always more prominent in the K₄A and K₄N fibrils than in the WT fibrils in at least six experiments.