SIGNAL TRANSDUCTION CORE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该核心的目的是为有兴趣研究细胞转导和信号通路的研究人员提供设计，制作表达荧光融合蛋白的载体，用于细胞内定位和蛋白质运输的技术援助。虽然分子生物学的使用是必不可少的，但细胞生物学研究提供了连接体外和体内数据的纽带，有助于证实提出的假设或模型。荧光标记蛋白质的研究结果通常显示蛋白质的稳态分布，但它们不能直接提供有关分子动力学性质的信息。为了确定蛋白质在体内的动力学性质，必须使感兴趣的蛋白质的运动可见。最常用的技术是FRAP（光漂白后荧光恢复）。在这种方法中，使用高强度激光脉冲快速漂白细胞的一小块区域。来自邻近区域的未漂白分子的运动，然后通过延时显微镜记录为漂白区域中荧光的恢复。显微镜还允许延时电影字幕监测细胞中荧光标记的蛋白质运动，以响应不同的药物治疗改变特定途径，配体受体下游目标等。体内分子相互作用也可以通过荧光能量转移（FRET）来研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this core is to provide technical assistance in designing, making vectors expressing fluorescent fusion proteins for intracellular localization and protein trafficking to investigators interested in studying cellular transduction and signaling pathways. Although the use of molecular biology is indispensable, cell biology studies offer a link that connect in vitro and in vivo data and is useful to confirm hypothesis or models proposed. Observations from studies with fluorescently tagged proteins typically show the steady state distribution of a protein, but they do not directly provide information about the kinetic properties of molecules. To determine the kinetic properties of proteins in vivo, the movement of the protein of interest must be made visible. The most commonly used technique for this is FRAP (fluorescence recovery after photobleaching). In this method, a small area of a cell is rapidly bleached using a high-intensity laser pulse. The movement of unbleached molecules from the neighboring areas is then recorded by time-lapse microscopy as the recovery of fluorescence in the bleached area. The microscope will also allow for time lapse movie caption to monitor fluorescently labeled protein movement in the cell in response to different drug treatment altering a specific pathway, ligand-receptor downstream targets and so on. In vivo molecular interactions could also be investigated by fluorescent energy transfer (FRET).