A growing number of studies have used new generation technologies to characterize the protein constituents of cilia and centrosomes. This has led to the identification of a vast number of candidate ciliary or centrosomal proteins, whose subcellular localization needs to be investigated and validated. Here, we describe a simple and inexpensive method for analyzing the subcellular localization of candidate cilium- or centrosome-associated proteins, and we illustrate the utility as well as the pitfalls of this method by applying it to a group of ASH (ASPM, SPD-2, Hydin) domain-containing proteins, previously predicted to be cilia- or centrosome-associated proteins based on bioinformatic analyses.
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