Despite its advantages, FRET has some limitations:
Distance Dependency: FRET is highly dependent on the distance between the donor and acceptor fluorophores, which can limit its applicability to certain interactions. Photobleaching: Prolonged exposure to light can bleach the fluorophores, reducing the efficiency and accuracy of the measurements. Complex Data Analysis: The interpretation of FRET data can be complex and requires sophisticated software and expertise. Potential for Non-specific Interactions: Non-specific binding of fluorophores may lead to false-positive results, requiring careful experimental design and controls.
