Effect of solubilization temperature and increase in ultracentrifugation time on the sedimentation velocity profile of PK-resistant PrP^Sc and infectivity.

<p>Brain homogenates from <i>tg338</i> mice infected with LA21K <i>fast</i> prions were solubilized at 4°C (<b>A</b>; <b>C</b>) or 37°C (<b>B</b>), loaded on top of a linear optiprep gradient and ultracentrifuged for 45 min (<b>A</b>, <b>B</b>) or 90 min (<b>C</b>). Fractions collected from the gradient (numbered from top to bottom) were analyzed for PK-resistant PrP^Sc content (black line; left axis) and for infectivity (red line; right axis). The mean levels of PK-resistant PrP^Sc per fraction have been obtained from the immunoblot analysis of n≥3 independent fractionations. As the replicates gave consistent results, these data were combined and fit. Fraction infectivity was determined by a Rov cell assay. It is based on the quantification of PrP^Sc-containing Rov cells by immunofluorescence using PrP^Sc-specific antibodies. The cells were exposed in parallel to fraction aliquots and to serial tenfold dilutions of a LA21K <i>fast</i>-infected brain (expressed as relative infectious doses) prepared in the same conditions. The data presented are the mean ± SEM of n = 3 independent titrations. Data presented in A are from ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003702#ppat.1003702-Tixador1" target="_blank">[20]</a> and this study.