Figure 2.

Aggregate isolation protocol and validation with amyloid prion harbouring strains. (a) Aggregate isolation workflow. (b) Immunoblotting of the SDS-resistant fraction. Equal amounts of aggregates isolated from [RNQ⁺][psi⁻], [rnq⁻][psi⁻] and [RNQ⁺][PSI⁺] were re-suspended in SDS-sample buffer and split in two. One fraction was heated at 95°C for 5 min, while the other was left untreated. The samples were then resolved on SDS–PAGE and immunoblotted with an antibody against S. cerevisiae Rnq1. (c). Measured abundance of Rnq1 and Sup35 identified in the SDS-resistant and -soluble fraction of the isolated aggregates by mass spectrometry. Equal amount of aggregate isolated from [RNQ⁺][psi⁻], [rnq⁻][psi⁻] and [RNQ⁺][PSI⁺] were re-suspended in SDS-sample buffer and run through approximately 2.5 cm of a 4–15% TGX SDS–PAGE (Bio-Rad) prior to measurement by mass spectrometry. See Material and methods for further details. Expression values from total lysate across the compendium of yeast mass spectrometry experiments [19] are provided as a point of reference. (Online version in colour.)