Quantitative analysis of H-Ras localization using confocal microscopy

Protein prenylation is an essential post-translational modification, wherein an isoprenoid group is irreversibly attached to the C-terminus of approximately 2 % of the mammalian proteome. This modification facilitates membrane association and functional regulation of diverse signaling proteins, including Ras GTPases. Ras proteins are members of a large superfamily of small GTPases, and therefore disruptions in prenylation impact their localization and importantly contribute to pathologies including cancer and metabolic diseases. This chapter presents a robust, semi-automated workflow for quantifying the subcellular distribution of GFP-H-Ras in adherent epithelial cells, providing a reproducible platform for comparative studies of prenylation and isoprenoid analog effects. The procedure combines high-resolution fluorescence microscopy with image analysis to calculate raw integrated density as a metric of localization. This pipeline is scalable for high-content imaging datasets and adaptable to a variety of prenylated protein systems for mechanistic studies of protein membrane targeting.