Moerke Nathan J, Hoffman Gregory R
Harvard Medical School, ICCB-Longwood Screening Facility, Boston, Massachusetts.
Curr Protoc Chem Biol. 2011 Mar 1;3(1):39-52. doi: 10.1002/9780470559277.ch100153.
The in-cell western (ICW) technique is a cell-based immunoassay method for quantitative measurement of protein expression or phosphorylation levels that can be used for both small molecule and siRNA screening. The method involves growth of cells in microplates, fixation, permeabilization, and staining with specific antibodies and/or cell labeling dyes. ICW assays take advantage of the properties of near-infrared dyes to achieve higher signal-to-noise ratios than are possible for methods utilizing fluorophores in the visible range of the spectrum, and typically involve measurements using two fluorescent channels: one to measure levels of the target of interest, and one to measure total cell number for normalization. The ICW method is readily adaptable to high-throughput format and has been successfully used with a variety of targets and cell lines. The protocols in this unit describe an ICW procedure for quantitative measurement of rpS6-phosphorylation as an endpoint for monitoring mTORC1 signaling in HeLa cells. This assay can be used for small molecule or siRNA screening, and with modification is adaptable to other cell lines and targets. Curr. Protoc. Chem. Biol. 3:39-52 © 2011 by John Wiley & Sons, Inc.
细胞内western（ICW）技术是一种基于细胞的免疫测定方法，用于定量测量蛋白质表达或磷酸化水平，可用于小分子和siRNA筛选。该方法包括在微孔板中培养细胞、固定、通透处理，并用特异性抗体和/或细胞标记染料进行染色。ICW测定利用近红外染料的特性，以实现比使用光谱可见范围内荧光团的方法更高的信噪比，并且通常涉及使用两个荧光通道进行测量：一个用于测量目标物的水平，另一个用于测量总细胞数以便进行归一化。ICW方法易于适应高通量形式，并且已成功用于多种靶标和细胞系。本单元中的方案描述了一种ICW程序，用于定量测量rpS6磷酸化，作为监测HeLa细胞中mTORC1信号传导的终点。该测定可用于小分子或siRNA筛选，并且经过修改后可适用于其他细胞系和靶标。《化学与生物学实验指南》第3卷：39 - 52页 2011年 约翰威立国际出版公司版权所有
