Endogenous α-SYN protein analysis from human brain tissues using single-molecule pull-down assay

Abstract

Alpha-synuclein (-SYN) is a central molecule in Parkinsons disease pathogenesis. Despite several studies, the molecular nature of endogenous -SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of bio-specimens. Here, we introduce -SYN single-molecule pull-down (-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual -SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. A SiMPull assay enables us to count the number of immuno-precipitated proteins, and reveal the stoichiometry of protein complexes by single-molecule fluorescence imaging. This powerful assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimers disease and Parkinsons disease.