Figure 1.

Diagrammatic representation of cell-free conversion assays. A small quantity of a brain homogenate containing PrP^Sc, usually at quantities lower than is normally detectable when immunoblotting, is added to a pool of “normally” folded host-encoded protein. This can be either purified recombinant proteins (recPrP) or an uninfected brain homogenate. Briefly, the assays undergo periods of incubation, whereby the PrP^Sc seeds can interact with recPrP and cause protein misfolding and aggregation. Intermittently, periods of sonication are used to break down larger aggregates to allow further conversion of recPrP to PrP^Sc. The assay products are then assessed using Western blot analysis. These systems result in a large accumulation of PrP^Sc, which have occurred in a cell-free system, and, as the result of protein-templated conversion directed from the initial extremely small quantity of PrP^Sc added to the reaction in the first instance.