Browsing Department of Medicine Faculty: Research and Presentations by Subjects
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Improved Immunodetection of Endogenous Î±-SynucleinÎ±-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.