Isolation and Analysis of Prion and Amyloid Aggregates from Yeast Cells.

Abstract

Amyloid fibers are large and extremely stable structures that can resist denaturation by strong anionic detergents, such as sodium dodecyl sulfate or sarkosyl. Here, we present two complementary analytical methods that exploit these properties, enabling the isolation and characterization of amyloid/prion aggregates. The first technique, known as semidenaturating detergent agarose gel electrophoresis, is an immunoblotting technique, conceptually similar to conventional western blotting. It enables the targeted identification of large detergent-resistant protein aggregates using antibodies specific to the protein of interest. The second method, called the technique for amyloid purification and identification, is a nontargeted approach that can isolate amyloid aggregates for analysis by tandem mass spectrometry. The latter approach requires no special genetic tools or antibodies, and can identify amyloid-forming proteins, such as prions, as well as proteins tightly associated with amyloid, from a variety of cell sources.