Figure 3: Fluorescence Recovery after Photobleaching (FRAP). FRAP is a useful technique to study the exchange of fluorescently tagged soluble sarcomeric proteins with their counterparts in the myofibril in living cells, processes referred to as protein dynamics. (a) In a FRAP experiment, the construct containing the studied protein linked to fluorescence protein was transfected and expressed in the target cells. FRAP experiment can be setup in three simple steps. First, pre-bleach Image was recorded using low intensity light as control. Second, an interested region was chosen and bleached using high intensity laser light. Third, a series of images were recorded using low light intensity to follow the fluorescence intensity change during recovery. (b) When the fluorescently tagged protein was expressed in the cell, most proteins were localized in the some special structure or area, but there are also free proteins in the cytoplasm pool. The protein in the special structure was in a dynamic exchange with the cytoplasm pool. By applying high intensity laser beam in a small area, the fluorescence protein in the region of interested was irreversibly bleached due to the photochemical destruction of the fluorophore, but the studied protein was intact and still in exchange with free fluorescently tagged proteins in the cytoplasmic pool. This results in the bleached protein coming out of the myofibril, and unbleached fluorescently labeled protein coming into the bleached region, and thus the fluorescence in recovered in the initially photobleached region.