Development of in-cell Western assays using far-red fluorophores

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.