Yano Yoshiaki, Kawano Kenichi, Omae Kaoru, Matsuzaki Katsumi
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Methods Enzymol. 2012;504:355-70. doi: 10.1016/B978-0-12-391857-4.00018-5.
Tag-probe labeling methods have advantages over conventional fusion with fluorescent proteins in terms of smaller labels, surface specificity, availability of pulse labeling, and ease of multicolor labeling. With this method, the gene of the target protein is fused with a short tag sequence, expressed in cells, and the protein is labeled with exogenous fluorescent probes that specifically bind to the tag. Various labeling principles, such as protein-ligand interaction, peptide-peptide interaction, peptide-metal interaction, and enzymatic reactions, have been applied to the tag-probe labeling of membrane receptors. We describe our coiled-coil tag-probe method in detail, including the design and synthesis of the tag and probe, labeling procedures, and observations by confocal microscopy. Applications to the analysis of receptor internalization and oligomerization are also introduced.
标签-探针标记方法相对于传统的荧光蛋白融合方法具有优势，体现在标记更小、表面特异性、脉冲标记的可行性以及多色标记的简便性等方面。通过这种方法，将目标蛋白的基因与一个短标签序列融合，在细胞中表达，然后用能特异性结合该标签的外源荧光探针标记蛋白质。各种标记原理，如蛋白质-配体相互作用、肽-肽相互作用、肽-金属相互作用和酶促反应，已应用于膜受体的标签-探针标记。我们详细描述了我们的卷曲螺旋标签-探针方法，包括标签和探针的设计与合成、标记程序以及共聚焦显微镜观察。还介绍了其在受体内化和寡聚化分析中的应用。
