This protocol describes the technique of Fast Relaxation Imaging (FReI) as applied to protein folding inside living cells. The required modifications of a fluorescence microscope by addition of a diode laser temperature jump source, a yellow/blue switchable light-emitting diode source, and a two-color CCD camera to collect movies of protein dynamics inside cells are discussed. A description of how proteins are labeled for imaging, how cells are prepared for imaging, and how imaging of kinetics inside cells with millisecond time resolution is obtained, along with the complementary in vitro experiments, is also provided. The ability to carry out comparative in vitro and "in-cell" measurements on the same setup al... More
This protocol describes the technique of Fast Relaxation Imaging (FReI) as applied to protein folding inside living cells. The required modifications of a fluorescence microscope by addition of a diode laser temperature jump source, a yellow/blue switchable light-emitting diode source, and a two-color CCD camera to collect movies of protein dynamics inside cells are discussed. A description of how proteins are labeled for imaging, how cells are prepared for imaging, and how imaging of kinetics inside cells with millisecond time resolution is obtained, along with the complementary in vitro experiments, is also provided. The ability to carry out comparative in vitro and "in-cell" measurements on the same setup allows for direct comparison of the features distinguishing cellular protein folding (or other biomolecular processes) from studies performed in dilute solution.
