AbstractÎ±-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative Î±-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous Î±-synuclein. This difficulty has largely restricted the progress for Î±-synucleinopathy research. Here, we report that Î±-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous Î±-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous Î±-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous Î±-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous Î±-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous Î±-synuclein, thereby facilitating Î±-synuclein research.
CitationPLoS One. 2011 Aug 19; 6(8):e23939
- Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System.
- Authors: Preterre C, Corbillé AG, Balloy G, Letournel F, Neunlist M, Derkinderen P
- Issue date: 2015
- Sensitive western blotting for detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and extracellular spaces.
- Authors: Sasaki A, Arawaka S, Sato H, Kato T
- Issue date: 2015 Sep 18
- A new method for quantitative immunoblotting of endogenous α-synuclein.
- Authors: Newman AJ, Selkoe D, Dettmer U
- Issue date: 2013
- Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral-mediated RNAi.
- Authors: Sapru MK, Yates JW, Hogan S, Jiang L, Halter J, Bohn MC
- Issue date: 2006 Apr
- alpha-Synuclein is phosphorylated in synucleinopathy lesions.
- Authors: Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio K, Iwatsubo T
- Issue date: 2002 Feb