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.
Highlights
Introduction aSynuclein (a-syn) is a small soluble protein (17 kDa) that contains 140 amino acids and is predominantly expressed in the brain. a-Syn localizes to synaptic vesicles and the nucleus— the name ‘‘synuclein’’ [1,2]
A-syn is predominantly expressed in nervous system tissues [1], it is difficult to detect endogenous a-syn in cultured cell lines by immunoblotting
We recently demonstrated that endogenous a-syn is undetectable in human neuroblastoma SHSY5Y cells, but detectable in human melanoma cells such as SKMEL28 [11]
Summary
Introduction aSynuclein (a-syn) is a small soluble protein (17 kDa) that contains 140 amino acids and is predominantly expressed in the brain. a-Syn localizes to synaptic vesicles and the nucleus— the name ‘‘synuclein’’ [1,2]. Synuclein (a-syn) is a small soluble protein (17 kDa) that contains 140 amino acids and is predominantly expressed in the brain. A-Syn localizes to synaptic vesicles and the nucleus— the name ‘‘synuclein’’ [1,2]. The exact function of a-syn is still unclear, substantial evidence exists to suggest that this protein is predominantly natively unfolded in solution but can bind to phospholipid membranes by adopting an a-helical secondary structure in its N-terminal region [3,4]. Asyn regulates the pool of synaptic vesicles through an interaction with membranes [2]. The loss appears to be caused by neuronal cell death due to toxicity of accumulated a-syn [2]. Much effort has been directed toward investigating the a-syn molecule biochemically as well as immunohistochemically
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