These probes are designed to detect the accumulation of phosphoinositides at the plasma membrane. From the N-terminus, the probes consist of CFP, PH domain, linker, YFP, and a membrane-targeting signal. The PH domain is selected on the basis of affinity to the phosphoinositides to be detected. In case of a probe for PIP3, the PH domain of GRP is used. The membrane-targeting signal is chosen so that the probe is delivered to the desired place. We often use the C-terminal domain of KRas protein, which delivers probes efficiently to the plasma membrane. The mode of action is explained with this probe for PIP3. When the PIP3 level at the plasma membrane is low, the PH domain floats in the cytoplasm, in which condition FRET efficiency is expected to be low. When PIP3 accumulates at the plasma membrane, the PH domain is fixed to the plasma membrane, which brings CFP in close proximity of YFP and, thereby, increasing the FRET.
