Analysis of Nucleosome Structure in Polyacrylamide Gel by the Förster Resonance Energy Transfer Method

Resumo

A technique for analyzing the structure of (Cy3, Cy5)-labeled nucleosomes in polyacrylamide gel after electrophoresis under native conditions was developed based on the Förster resonance energy transfer (FRET) effect. It has been shown that the correct application of this technique requires monitoring of nonspecific intermolecular FRET and fluorescence reabsorption. A comparative analysis of the results of the FRET measurements of two types of nucleosomes and their complexes with yeast protein FACT was performed, which confirmed the similarity of the structural features of nucleosomes detected in the gel and in aqueous solution. Application of FRET analysis in combination with electrophoresis makes it possible not only to separate, visualize components of a complex mixture, and to evaluate their relative content but also to characterize the structural differences between these complexes in situ.