Detection of prion protein oligomers by single molecule fluorescence imaging.
Detection of prion protein oligomers by single molecule fluorescence imaging.
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通过单分子荧光成像检测朊病毒蛋白寡聚体。
DOI：
10.1111/j.1440-1789.2012.01316.x
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发表时间：
2013
期刊：
影响因子：
2.3
通讯作者：
Iwaki T
中科院分区：
文献类型：
--
作者：
Shibano S;Sasaki K;Kidoaki S;Iwaki T
PrP的聚合度与朊病毒疾病的病理机制有着密切的关系。我们使用全内反射荧光显微镜 (TIRFM) 在分子水平上检查了朊病毒感染细胞裂解物中 PrP 的聚合状态。粗裂解物根据分子大小通过凝胶过滤旋转柱进行分级。使用荧光素标记的抗 PrP 抗体（mAb SAF70 和 mAb 8G8）探测富含寡聚体和富含单体的级分。检测到不同强度的荧光点，其中使用 mAb SAF70 的寡聚物级分样品中强荧光点的比例高于使用 8G8 的寡聚物级分样品，其特定表位被认为埋藏在异常 PrP 分子中。结果表明，可以特异性地检测到PrP寡聚物，并观察到异常PrP分子的构象变化。 TIRFM 成像可能有助于确定病理过程中 PrP 低聚物的聚合状态和性质。
The degree of polymerization of PrP has a close relationship with the pathological mechanisms of prion diseases. We examined, at the molecular level, the polymerization state of PrP in lysates of prion‐infected cells using total internal reflection fluorescence microscopy (TIRFM). The crude lysates were fractionated by gel‐filtration spin columns according to their molecular size. Both the oligomer‐rich and the monomer‐rich fractions were probed with fluorescein‐labeled anti‐PrP antibodies (mAb SAF70 and mAb 8G8). Fluorescent spots of varying intensity were detected, with the ratio of intense fluorescent spots being greater in the oligomer fraction samples with mAb SAF70 than those with 8G8, the specific epitope of which is thought to be buried in abnormal PrP molecules. The results indicated that PrP oligomers could be specifically detected and conformational changes of abnormal PrP molecules observed. Imaging by TIRFM may aid in determining the polymerization state and properties of PrP oligomers in pathological processes.