Abstract
This chapter describes the basic methodology used to systematically determine the subcellular localization of novel human proteins, as they have been derived from past and current cDNA sequencing projects worldwide. Since, localization is wholly dependent upon targeting sequences within the protein of interest, tagging of a protein with green fluorescent protein (GFP) always carries the risk that these targeting sequences become masked, which will finally lead to a mislocalization of the tagged protein. The localization that best matches the bioinformatic data is then considered as the correct one. Finally, data are verified by colocalization of the GFP fusion proteins with established endogenous organelle-specific markers. The localization that best matches the bioinformatic data is then considered as the correct one. Finally, data are verified by colocalization of the GFP fusion proteins with established endogenous organelle-specific markers. In parallel cultures or following image acquisition from live cells, it is important to fix the cells and remove any soluble GFP signal that may be obscuring more subtle localization patterns.
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