Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells.
Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells.
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DOI：
10.21769/bioprotoc.4614
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发表时间：
2023-02-20
期刊：
影响因子：
0.8
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中科院分区：
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细胞表面组在生理学、发育生物学和疾病状态等方面都至关重要。蛋白质及其在细胞膜上的调节机制的精确鉴定一直具有挑战性，并且通常使用共聚焦显微镜、双光子显微镜或全内反射荧光显微镜（TIRFM）来确定。其中，TIRFM是最精确的，因为它利用在具有不同折射率的两个表面的界面处产生空间限定的倏逝波。渐逝波的有限穿透照亮了狭窄的样本场，这有利于荧光标记的蛋白质在细胞膜上而不是在细胞内的定位。除了限制图像的深度，TIRFM还显著提高了信噪比，这在活细胞的研究中特别有价值。在这里，我们详细介绍了HEK 293-T细胞中光遗传学激活的蛋白激酶C-ε的RTIRFM分析方案，以及数据分析，以证明该构建体在光遗传学激活后易位到细胞表面。图形摘要
The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices. The limited penetration of the evanescent wave illuminates a narrow specimen field, which facilitates the localization of fluorescently tagged proteins at the cell membrane but not inside of the cell. In addition to constraining the depth of the image, TIRFM also significantly enhances the signal-to-noise ratio, which is particularly valuable in the study of live cells. Here, we detail a protocol for micromirror TIRFM analysis of optogenetically activated protein kinase C-ε in HEK293-T cells, as well as data analysis to demonstrate the translocation of this construct to the cell-surface following optogenetic activation. Graphic abstract