High-throughput quantification of posttranslational modifications in situ by CA-FLIM

Signal transduction is mediated by posttranslational modifications (PTMs) of proteins in complex signaling networks. Quantifying PTM levels of multiple network components in response to a stimulus is therefore the key to understand how their concerted activities give rise to cellular function. We have shown that fluorescence lifetime imaging microscopy (FLIM) on cell arrays (CA-FLIM) provides a method to accurately quantify PTM levels of many proteins in situ. Herein, we describe the detailed protocol for CA-FLIM. Less than 2 days are needed from cell array preparation to data analysis, where the main limiting step is the 24h needed for transfection. After generating a single cell array containing 384 spots, it can be imaged and analyzed in less than 2h.