title: Improved immunodetection of endogenous α-synuclein.
abstract: α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-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 α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-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 α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-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 α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.
authors: Department of Medicine, Center for Molecular Chaperone/Radiobiology and Cancer Virology, Georgia Health Sciences University, Augusta, Georgia, United States of America.
authors: Byung Rho
authors: BR
authors: Lee
authors: Tetsu
authors: T
authors: Kamitani
mesh_terms: D000818:Animals; D002460:Cell Line; D005557:Formaldehyde; D006801:Humans; D015151:Immunoblotting / Q000379:methods* / Q000592:standards; D051379:Mice; D010766:Phosphorylation; D011108:Polymers; D014018:Tissue Distribution; D051844:alpha-Synuclein / Q000032:analysis*
chemical_list: D011108:Polymers; D051844:alpha-Synuclein; D005557:Formaldehyde; C003043:paraform