Determination of FRET Efficiency Using Donor Photobleaching Method with Green Fluorescent Proteins as Energy Transfer Pair

Abstract:

      In this work, two new green fluorescence proteins, named MiCy and mKo, were used as the energy transfer pair and their FRET efficiency was measured by the donor photobleaching method. The excitation and emission spectra and the photobleaching curves of the purified MiCy and mKo proteins were determined. The results suggested that MiCy is the photobleaching donor and that mKo is a photostable acceptor. By using Ni -NTA -agarose as the FRET model, true transfer efficiency between MiCy and mKo was successfully detected on Confocal microscope. In conclusion, the MiCy - mKo is a proper FRET pair, and the donor photobleaching method can provide a very specific measurement of FRET efficiency.