Fig. 9
Assessment of the capacity of prion-coated glass spheres to seed PMSA reactions and confirmation of the adsorption of recombinant prions onto the glass surface. After a 24 h PMSA reaction, clean glass spheres of 9–13 µm diameter were seeded with Ust02 recombinant prions. The spheres potentially coated with recombinant prions were allowed to settle and separated from the PMSA-product supernatant. While the supernatant was directly used as seed, serially diluted (from 10–1 to 10–8) in new PMSA substrate complemented with 1 mm zirconia silicate beads, submitting the tubes to a single 24 h PMSA round, the sediment of glass spheres was thoroughly washed with PBS and finally re-suspended in the same volume of Conversion buffer (devoid of rec-PrP) as the original reaction. These solutions with re-suspended glass spheres were serially diluted in the same way as done previously with the PMSA product supernatant. The serial dilutions of the glass sphere suspension were used as seed in the new PMSA substrate supplemented with zirconia silicate beads, and a single 24 h PMSA round was conducted. Results of the PMSA comparing the seeding capacity of PMSA supernatant and the suspension of prion-coated glass spheres, were monitored by proteinase K (PK) digestion, electrophoresis, and total protein staining. In both cases, rec-PrPres was detected in tubes seeded with dilutions 10–6 of PMSA product or sphere suspension, showing similar seeding abilities and demonstrating that glass spheres are indeed coated with recombinant prions that conserve their seeding capacity. Additionally, the prion-coated glass sphere suspension used as seed was also digested directly with proteinase K after extensive washing. Once digested, the glass spheres were re-suspended in loading buffer (NuPage 4 × ) and the resulting supernatant, excluding the glass spheres, was loaded into an acrylamide gel and stained for total protein. As shown in the gel below, the prions released from the glass spheres after digestion show the expected electrophoretic pattern for Ust02 prions, confirming their adsorption to the glass surface. MW: Molecular weight marker