Department of Medicine, Center for Molecular Chaperone/Radiobiology and Cancer Virology, Georgia Health Sciences University, Augusta, Georgia, United States of America.
PLoS One. 2011;6(8):e23939. doi: 10.1371/journal.pone.0023939. Epub 2011 Aug 19.
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.
α-突触核蛋白是理解神经退行性α-突触核蛋白病（如帕金森病）发病机制的关键分子。然而，尽管进行了广泛的研究，但其确切功能仍不清楚，部分原因是内源性α-突触核蛋白的免疫印迹检测存在困难。这种困难在很大程度上限制了α-突触核蛋白病研究的进展。在这里，我们报告说α-突触核蛋白单体容易从印迹膜上轻易脱落，导致无法或非常差的检测。为了防止这种脱落，我们将印迹膜用多聚甲醛进行轻度固定，应用于免疫印迹法。令人惊讶的是，这种固定导致内源性α-突触核蛋白的清晰和强烈检测，这是常规免疫印迹法无法检测到的。具体来说，我们能够在各种人类细胞系（包括 SH-SY5Y、HEK293、HL60、HeLa、K562、A375 和 Daoy）以及一种小鼠细胞系 B16 和几种小鼠组织（如脾和肾）中检测到内源性α-突触核蛋白。此外，值得注意的是，我们能够在几种人类细胞系中清晰地检测到磷酸化丝氨酸 129 的内源性α-突触核蛋白。因此，在一些组织和培养细胞中，简单地固定印迹膜就可以使内源性α-突触核蛋白变得容易检测。这种改进的免疫印迹法将使我们能够检测以前无法检测到的内源性α-突触核蛋白，从而促进α-突触核蛋白的研究。
