Miyashita Toshiyuki
Department of Genetics, National Research Institute for Child Health and Development, Tokyo, Japan.
Methods Mol Biol. 2004;261:399-410. doi: 10.1385/1-59259-762-9:399.
Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here I describe the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins. Ideally, the intracellular co-localization of the two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence, or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies after transient transfection. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein.
共聚焦激光扫描显微镜是观察完整细胞内蛋白质共定位的最佳方法。由于采用点扫描/针孔检测系统，可消除样本中扫描点附近区域的光干扰，从而实现高Z轴分辨率。使用灵敏的光电倍增管进行荧光检测，能够使用窄带通滤光片，使两个光谱之间的串扰（重叠）降至最低。这在通过多色标记展示蛋白质共定位时尤为重要。在此，我将描述检测瞬时表达的标签蛋白以及检测内源性蛋白的方法。理想情况下，应展示两种内源性蛋白的细胞内共定位。然而，当没有针对目标蛋白的抗体可用于免疫荧光检测，或者现有的细胞系表达目标蛋白的水平不足以进行免疫荧光检测时，另一种方法是用编码标签蛋白的表达质粒转染细胞，并在瞬时转染后用抗标签抗体对细胞进行染色。不过，需要注意的是，对目标蛋白进行标记或使其过表达可能会潜在地改变目标蛋白的细胞内定位或功能。
