Tracking individual membrane proteins using quantum dots

Single-particle tracking of individual membrane molecules is now the method of choice to decipher the molecular organization of the plasma membrane. By labeling proteins or lipids with latex beads, 40-nm gold nanoparticles, or small organic fluorophores, it is possible to analyze the mechanisms controlling their lateral dynamics. Semiconductor quantum dots (QDs) provide several advantages for tracking membrane molecules: (1) Their size, which is intermediate between those of organic dyes (1-4 nm) and large beads (100 nm to 1 µm), remains close to the molecular scale; (2) their photostability allows observation over long durations; (3) parallel detection of multiple spots in a field of view is easy; and (4) multicolor imaging is facilitated by their absorption properties. In general, the labeling of membrane molecules is based on the targeting of an extracellular epitope by a tagged antibody or ligand. By progressively decreasing the concentration of markers, a regime is reached where isolated tags can be detected and tracked. We present here a protocol based on the successive use of biotinylated primary antibodies and streptavidin-coated QDs.