HOW DO WE KNOW?

FIG. 5.8

Do proteins move in the plane of the membrane?

BACKGROUND Fluorescent recovery after photobleaching (FRAP) is a technique used to measure mobility of molecules in the plane of the membrane. A fluorescent dye is attached to proteins embedded in the cell membrane in a process called labeling. A laser is then used to bleach a small area of the membrane.

HYPOTHESIS If membrane components such as proteins move in the plane of the membrane, the bleached spot should become fluorescent over time as unbleached fluorescent molecules move into the bleached area. If membrane components do not move, the bleached spot should remain intact.

EXPERIMENT AND RESULTS

FIG. 5.8

Photo source: FRAP of cytoplasmic EGFP in living HeLa cells, performed using an UltraVIEW® spinning disk confocal system (PerkinElmer Inc.). HeLa cells were transfected with pEGFP-C1 (Clontech Laboratories, Inc.) using GeneJuice® transfection reagent (Novagen®). A region of interest in the cytoplasm was photobleached using the UltraVIEW® photokinesis unit, and the recovery of fluorescence in this region was observed. (Fluorescence Recovery After Photobleaching (FRAP) using the UltraVIEW PhotoKinesis accessory, PerkinElmer Technical Note).

CONCLUSION The gradual recovery of fluorescence in the bleached area indicates that proteins move in the plane of the membrane.

SOURCE Peters, R., et al. 1974. “A Microfluorimetric Study of Translational Diffusion in Erythrocyte Membranes.” Biochim Biophys Acta 367:282–294.