Abstract
Site-specific labeling of cellular proteins with chemical probes is a powerful tool for live cell imaging of biological processes. One popular system, known as the SNAP-tag, is based on an engineered variant of the 20-kDa DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (AGT) that covalently reacts with O(6)-benzylguanine (BG) and can be derivatized with a number of reporter groups. For studying the endocytosis and recycling of cell surface proteins, the covalent nature of BG binding to the SNAP-tag is problematic, since removing excess noninternalized probe from the cell surface is not feasible. Here we describe a modification of the SNAP-tag technology that permits the rapid release of fluorescently labeled probes from the cell surface without affecting the population of labeled molecules sequestered within endosomes. This simple yet effective approach allows quantitative measurements of endocytosis and recycling in both imaging and biochemical assays and is especially useful when studying endosomal dynamics in live cells.