Imaging protein kinase Cα activation in cells
Imaging protein kinase Cα activation in cells
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DOI：
10.1126/science.283.5410.2085
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发表时间：
1999-03-26
期刊：
影响因子：
56.9
通讯作者：
Parker, PJ
中科院分区：
文献类型：
--
作者：
Ng, T;Squire, A;Parker, PJ
通过荧光寿命成像显微镜（FLIM）测量的空间分辨荧光共振能量转移（FRET），为追踪活细胞培养物中荧光标记蛋白质的催化活性提供了一种方法，并能够确定固定细胞和组织中蛋白质的功能状态。在这里，蛋白激酶C α (PKC α)激活的动态标记物被识别和利用。通过结合荧光标记的磷酸化位点特异性抗体检测PKC α的激活；由此产生的FRET通过供体荧光团在PKC α上通过FLIM测量。该方法能够在活细胞和固定培养细胞中成像PKC α活化，也适用于病理样本。
Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase C alpha (PKC alpha) activation is identified and exploited. Activation of PKC alpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKC alpha by FLIM. This approach enabled the imaging of PKC alpha activation in live and fixed cultured cells and was also applied to pathological samples.