Immunofluorescence of Intracellular Targets in Live Cells

Abstract

Every second, living human cells are executing complex functions to initiate or influence various processes such as energy production, growth, metabolism and reproduction. This also means that intracellular structures are rapidly moving and morphing as the various proteins and organelles interact. Two important goals for scientists attempting to understand and manipulate intracellular processes are to visualize structural movements within the cell and to use specifically generated proteins to adjust interactions within the cell. Both aims could be achieved if the desired proteins were able to cross through the cell membrane lipid bilayer. Currently, the methods for delivering proteins into live cells can compromise the integrity of the cell’s defenses, are very inefficient to use or they rely on endocytosis, a natural process that ends in the degradation of many proteins. Recently, members of the University of Washington’s Gao Lab have made a breakthrough by creating a cholesterol tag that aids in delivery of proteins across the cell membrane. In this study, a second-generation hydrophobic tag was tested for functionality in delivery of single domain antibodies, Fab fragments, and full-sized antibodies into live cells. Better understanding the functions and limits of this cytosolic-delivery method will increase the accessibility of live-cell fluorescent imaging and open new possibilities in the field of intracellular protein therapies.