Detection and Localization of Type II Membrane Proteins by Endogenous Gene Tagging and Confocal Microscopy.

Type II membrane proteins are single-pass transmembrane proteins distinguished by their N-terminus facing the cytoplasmic side and C-terminus oriented toward the extracellular or luminal side. The localization of type II membrane proteins is crucial for their functional roles, molecular interactions, and biological activity. Studying their dynamics remains challenging due to limited availability of highly specific antibodies and potential artifacts introduced by overexpression systems. In this context, we outline a detailed protocol for monitoring the endogenous localization of type II membrane proteins in mammalian cells. Here, we combine CRISPR/Cas9-mediated endogenous protein tagging with confocal microscopy. This method provides a powerful tool for precisely labeling endogenous proteins and investigating the localization and dynamics of membrane proteins under physiological conditions.