Fig. 7

Evaluating the impact of bead movement on spontaneous misfolding efficiency of recombinant PrP by PMSA. A In order to assess whether bead movement is required along with a minimum available glass surface, glass tubes without beads were used instead of regular plastic tubes in the PMSA reaction. The internal surface of the glass tube is approximately 125π mm², equivalent to the surface of glass beads previously shown to induce spontaneous misfolding from the first PMSA round. The standard plastic tubes complemented with glass beads to achieve a surface area of 125π mm² were used as a positive control, while the glass tubes without beads served as negative control. Two independent experiments (E1 and E2) were performed for each condition, with two replicate tubes each, all subjected to a single 24 h PMSA round. Detection of rec-PrP^res through proteinase K digestion, electrophoresis and total protein staining revealed that the presence of glass beads, even within the glass tube, was necessary for efficient misfolding induction. B To further investigate the potential significance of bead movement in spontaneous misfolding, 1mm diameter glass beads were embedded in a plastic resin to immobilize them while exposing most of their surface to the PMSA substrate. Four reaction tubes were prepared with resin-embedded beads, accompanied by two tubes without beads and two tubes with the same amount of non-embedded glass beads. All tubes were subjected to a single 24 h PMSA round. Analysis following proteinase K digestion, electrophoresis, and total protein staining, showed no detectable rec-PrP^res in the tubes without beads or in the tubes with beads embedded in resin. This demonstrates that bead movement is also critical for efficient spontaneous misfolding in PMSA