Knowledge of protein localization within the cellular environment is critical for understanding the function of the protein and its regulatory networks. Protein localization data, however, have traditionally been accumulated from single or small-scale experiments. Transfected-cell arrays (TCAs) represent a robust alternative for the high-throughput analysis of gene/protein functions in mammalian cells. For protein localization studies, TCAs not only allow for the transfection and expression of over 1,000 genes in a single experiment but also make it feasible for simultaneous co-localization analyses of different subcellular compartments. In this chapter, we have described a protein co-localization protocol using transfected human cell arrays for a large set of cellular compartments, including the nucleus, ER, Golgi apparatus, mitochondrion, lysosome, peroxisome, and the microtubules, intermediate filaments and actin filaments. The application of these "organelle-co-localized cell arrays" facilitates the precise determination of the localizations of numerous recombinant proteins in a single experiment, making it currently the most efficient technique for high-throughput protein co-localization screening.
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