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.
Highlights
The possibility that Ser129-phosphorylation modulates the metabolic fate and toxicity of α -synuclein, the physiological and pathological roles of Ser129-phosphorylation remain unclear
Western blotting with anti-human α -synuclein monoclonal antibody (211) demonstrated that the signals for total α -synuclein monomers were enhanced by membrane fixation with paraformaldehyde in a concentration dependent manner, similar to the effects observed for LB509 antibody (Fig. 1A, Supplementary figure S1)
To assess sensitivity to detect α -synuclein monomers by membrane fixation, we investigated the signals resulting from western blotting using purified recombinant α -synuclein proteins that were partially phosphorylated by incubation with casein kinase 2 (CK2)
Summary
The possibility that Ser129-phosphorylation modulates the metabolic fate and toxicity of α -synuclein, the physiological and pathological roles of Ser129-phosphorylation remain unclear. Lee and Kamitani reported a simple but effective improvement in the western blotting technique and demonstrated that endogenous levels of total α -synuclein monomers, including phosphorylated and non-phosphorylated forms, could be detected in cell lines and mouse tissues by fixing the transferred membrane with 0.4% paraformaldehyde[11]. They observed that paraformaldehyde fixation blocked detachment of the target protein from the transferred membrane during incubation, resulting in signal enhancement[11]. The observed signal enhancement was seen in both α -synuclein monomers and other proteins, despite variations in molecular size, the effectiveness and optimal concentration of paraformaldehyde or glutaraldehyde differed for each protein or primary antibody
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