The ability to specifically label proteins within living cells can provide important information about protein dynamics and function. Here we use a technology to covalently tether fluorophores with different wavelengths to the specially designed HaloTag® reporter protein. We have achieved surface expression of the HaloTag® reporter protein by fusing it to a truncated integrin. In addition, we have developed a novel membrane-impermeant fluorophore. By using differently colored cell-impermeant and permeant fluorophores, we show spatial separation of membrane and intracellular protein pools, respectively. The truncated integrin protein, labeled with distinguishable fluorophores, can be followed in real time to study translocation of surface and intracellular protein pools. Using a HaloTag®-integrin fusion protein, we have shown the HaloTag® technology to be a powerful tool to study spatial separation and real-time translocation of protein pools in live cells.
Promega Notes 95, 16–19.