Improved Immunodetection of Endogenous α-Synuclein

alpha-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative alpha-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 alpha-synuclein. This difficulty has largely restricted the progress for alpha-synucleinopathy research. Here, we report that alpha-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 alpha-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous alpha-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 alpha-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous alpha-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous alpha-synuclein, thereby facilitating alpha-synuclein research.