Analytical ultracentrifugation as a tool in the studies of aggregation of the fluorescent marker, Enhanced Green Fluorescent Protein

Enhanced green fluorescent protein (EGFP) is a fluorescent marker used in bio-imaging applications, including as an indicator of folding or aggregation of a fused partner. However, the limited maturation, low folding efficiency, and presence of non-fluorescent states of EGFP can influence the interpretation of experimental data. To measure aggregation associated with de novo folding of EGFP from a high GdnHCl concentration, the analytical ultracentrifugation method was used. Absorption detection at 280 nm allowed to monitor the presence of monomers and aggregated forms. Fluorescence detection enabled the observation of only properly folded molecules with a functional chromophore. The results showed intensive aggregation of EGFP in low concentrations of GdnHCl with a continuous distribution of aggregated forms. The properly folded monomers with mature chromophore were fluorescent, while the conglomerates of EGFP molecules were not. These facts are essential for a proper interpretation of data obtained with EGFP labelling.