Sensitive western blotting for detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and extracellular spaces.
Sensitive western blotting for detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and extracellular spaces.
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DOI：
10.1038/srep14211
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发表时间：
2015-09-18
影响因子：
4.6
通讯作者：
Kato T
中科院分区：
文献类型：
--
作者：
Sasaki A;Arawaka S;Sato H;Kato T
α-突触核蛋白沉积在路易体，是帕金森病(PD)的病理标志，在丝氨酸129(Ser129)高度磷酸化。相比之下，正常大脑中很少有丝氨酸129磷酸化的α-突触核蛋白。这种差异提示Ser129-磷酸化参与了帕金森病的神经退行性变过程。然而，这一修改的作用尚不清楚。限制相关生化分析的一个因素是，蛋白质印迹法很难检测到内源性Ser129磷酸化的α-突触核蛋白，因为α-突触核蛋白单体在孵育过程中从转移膜上分离出来。在这里，我们报道了用4%多聚甲醛和0.01 ~ 0.1%戊二醛联合固定转移膜，使Ser129磷酸化α-突触核蛋白单体的灵敏度增加了约10倍，即使在条件培养液、人脑脊液、细胞系和人脑提取液中也能检测到内源性蛋白质。这种方法可以在生理和病理条件下对α-突触核蛋白在细胞内外空间之间的传递进行更详细的生化分析。
α-Synuclein deposited in Lewy bodies, a pathological hallmark of Parkinson’s disease (PD), is highly phosphorylated at serine 129 (Ser129). In contrast, there is very little Ser129-phosphorylated α-synuclein in the normal brains. This difference suggests that Ser129-phosphorylation is involved in neurodegenerative processes of PD. However, the role of this modification remains unclear. One limiting factor for relevant biochemical analyses is that it is difficult to detect endogenous Ser129-phosphoryated α-synuclein by western blotting, because α-synuclein monomers detached from the transferred membrane during incubation. Here, we reported that combination fixation of the transferred membrane with 4% paraformaldehyde and 0.01 ~ 0.1% glutaraldehyde produced an approximately 10-fold increase in the sensitivity for Ser129-phosphorylated α-synuclein monomers, allowing detection of endogenous proteins even in conditioned medium, human cerebrospinal fluid, and extracts from cell lines and human brain. This method may enable more detailed biochemical analyses for α-synuclein transmission between intra and extracellular spaces under physiological and pathological conditions.