使用双水相系统提取蛋白质分离高纯度细胞外囊泡
Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System.
作者信息
Kim Jongmin, Shin Hyunwoo, Kim Jiyoon, Kim Junho, Park Jaesung
机构信息
Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Gyeong-buk, Republic of Korea.
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeong-buk, Republic of Korea.
出版信息
PLoS One. 2015 Jun 19;10(6):e0129760. doi: 10.1371/journal.pone.0129760. eCollection 2015.
We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.
我们提出了一种简单快速的方法，通过使用聚乙二醇/葡聚糖水两相系统（ATPS）来分离细胞外囊泡（EVs）。该系统从EVs和血清蛋白的混合物中分离出超过约75%的黑色素瘤来源的EVs。为了提高EVs的纯度，采用了分批操作作为额外步骤以去除蛋白质污染物，并且去除了超过约95%的蛋白质污染物。我们还进行了逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（western blotting）以验证分离出的EVs的诊断适用性，并在分离出的EVs中检测到源自黑色素瘤细胞的信使核糖核酸（mRNA）和CD81。
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