Imaging protein kinase Calpha activation in cells.
Imaging protein kinase Calpha activation in cells.
复制标题
细胞内蛋白激酶 Cα 激活成像。
作者：
Tony Ng;A. Squire;G. Hansra;Frédéric Bornancin;Corinne Prevostel;A. Hanby;William A. Harris;Diana M Barnes;S. Schmidt;Harry Mellor;P. Bastiaens;Peter J. Parker
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）提供了一种用于追踪活细胞培养物内荧光标记蛋白质的催化活性的方法，并且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Calpha（PKCalpha）激活的动态标志物的识别和开发。PKCalpha的激活通过荧光标记的磷酸化位点特异性抗体的结合来检测;随后的FRET通过FLIM通过PKCalpha上的供体荧光团来测量。这种方法能够对活的和固定的培养细胞中的PKCalpha活化进行成像，并且也应用于病理样品。
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 Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.