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Analysis of amyloid aggregates using agarose gel electrophoresis.

Abstract：

:Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, SDS-stable aggregates and low-n oligomers of the Abeta peptide (Selkoe et al., 1982; Walsh et al., 2002) cause toxic effects associated with Alzheimer's disease (Selkoe, 2004). The discovery of prions in lower eukaryotes, for example, yeast prions [PSI(+)], [PIN(+)], and [URE3] suggested that prion phenomena may represent a fundamental process that is widespread among living organisms (Chernoff, 2004; Uptain and Lindquist, 2002; Wickner, 1994; Wickner et al., 2004). These protein structures are more stable than other cellular protein complexes, which generally dissolve in SDS at room temperature. In contrast, the prion polymers withstand these conditions, while losing their association with their non-prion partners. These bulky protein particles cannot be analyzed in polyacrylamide gels, because their pores are too small to allow the passage and acceptable resolution of the large complexes. This problem was first circumvented by Kryndushkin et al. (2003), who used Western blots of protein complexes separated on agarose gels to analyze the sizes of SDS-resistant protein complexes associated with the yeast prion [PSI(+)]. Further studies have used this approach to characterize [PSI(+)] (Allen et al., 2005; Bagriantsev and Liebman, 2004; Salnikova et al., 2005), and another yeast prion [PIN(+)] (Bagriantsev and Liebman, 2004). In this chapter, we use this method to assay amyloid aggregates of recombinant proteins Sup35NM and Abeta42 and present protocols for Western blot analysis of high molecular weight (>5 MDa) amyloid aggregates resolved in agarose gels. The technique is suitable for the analysis of any large proteins or SDS-stable high molecular weight complexes.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Bagriantsev SN,Kushnirov VV,Liebman SW

doi

10.1016/S0076-6879(06)12003-0

subject

Has Abstract

pub_date

2006-01-01 00:00:00

pages

33-48

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(06)12003-0

journal_volume

412

pub_type

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Analysis of amyloid aggregates using agarose gel electrophoresis.