Sensitive western blotting for detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and extracellular spaces

Abstract

α-Synuclein deposited in Lewy bodies, a pathological hallmark of Parkinson’s disease (PD), is highly phosphorylated at serine 129 (Ser129). In contrast, there is very little Ser129-phosphorylated α-synuclein in the normal brains. This difference suggests that Ser129-phosphorylation is involved in neurodegenerative processes of PD. However, the role of this modification remains unclear. One limiting factor for relevant biochemical analyses is that it is difficult to detect endogenous Ser129-phosphoryated α-synuclein by western blotting, because α-synuclein monomers detached from the transferred membrane during incubation. Here, we reported that combination fixation of the transferred membrane with 4% paraformaldehyde and 0.01 ~ 0.1% glutaraldehyde produced an approximately 10-fold increase in the sensitivity for Ser129-phosphorylated α-synuclein monomers, allowing detection of endogenous proteins even in conditioned medium, human cerebrospinal fluid, and extracts from cell lines and human brain. This method may enable more detailed biochemical analyses for α-synuclein transmission between intra and extracellular spaces under physiological and pathological conditions.