Abstract
Background
Rotor-Gene Q instrument was used to perform high-resolution protein thermal melt studies to characterize protein-small-molecule interaction. Fluorescent dye (1-anilino-8-naphthalenesulfonate (1,8-ANS)) is used as a reporter of protein unfolding to measure the protein melting temperature (T m). Variations in the fluorescence yield upon titration of small molecules with the protein resulted in poor melting curves at low gain while a high gain setting caused signal saturation leading to data loss.
Findings
Acquisition of data at multiple gains within a single experiment provided high-quality data for samples with both low and high fluorescence yields. The melting temperatures were measured for all the samples in one run, while avoiding loss of data due to signal saturation. This method was successfully used to measure the binding constant by titration of a small-molecule ligand with the target protein.
Conclusion
Protein thermal melt experiments using the Rotor-Gene Q instrument have been made feasible for samples that show variations in fluorescence yield. Furthermore, since protein melting is irreversible, using multiple gains in the same experiment prevented loss of sample and saved gain optimization time.