Fluorescence Recovery After Photobleaching (FRAP)
Fluorescence recovery after photobleaching (FRAP) is a versatile and widely accessible technique to investigate the kinetics of molecular in living cells, which has become an important tool for cell biologists.
FRAP experiment is performed under fluorescence microscopy in three steps. First, an initial fluorescence of fluorescent molecules is measured in the region of interest (ROI). Then, the fluorescent molecules are rapidly photobleached by focusing the high-intensity laser beam onto the defined area. Finally, the exchange of bleached molecules with unbleached molecules from the surrounding region is followed over time using a low-intensity laser. The time course of change in fluorescence intensity in the ROI after photobleaching is called FRAP curve, which yields information of both the recovery kinetics and the fraction of molecules free to diffuse.
Figure 1. FRAP
diagram of a same cell in time series
FRAP has gained high recognition for several features:
- Performed on most commercially available confocal setups without any special modifications.
- Decreasing the signal-to-noise ratio by focusing on a spot of a relatively small area and interrogating a limited number of fluorophores.
- Acquiring the ratio of mobile to immobile particles within the total population more easily compared to other techniques.
FRAP can be applied to characterize various phenomena:
- Measure the diffusion and fluidity of a membrane protein.
- Examine the diffusion of membrane-bound or soluble particles.
- Study the movement of particles between organelles.
- Observe intracellular trafficking of molecules through nuclear pores.
- Probe interactions between membrane proteins by geometry and stoichiometry.
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References
- Snapp E L, Altan N, Lippincott‐Schwartz J. Measuring protein mobility by photobleaching GFP chimeras in living cells. Current Protocols in Cell Biology. 2003, 19(1): 21.1. 1-21.1. 24.
- Van Royen M E, et al. Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells. The nucleus. Humana Press, Totowa, NJ, 2008: 363-385.
- Day C A, et al. Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP). Current Protocols in Cytometry. 2012, 62(1): 2.19. 1-2.19. 29.
