FACS-sorted particles reduce the data variance in optical tweezers-assisted dynamic force spectroscopy measurements

T Stangner, D Singer, C Wagner, C Gutsche, O Ueberschär, R Hoffmann and F Kremer
By combining optical tweezers-assisted dynamic force spectroscopy experiments with fluorescence activated cell sorting (FACS), we demonstrate a new approach to reducing the data variance in measuring receptor–ligand interactions on a single molecule level by ensuring similar coating densities. Therefore, the carboxyfluorescein-labelled monophosphorylated peptide tau226–240[pThr231] is anchored on melamine resin beads and these beads are sorted by FACS to achieve a homogeneous surface coverage. To quantify the impact of the fluorescence dye on the bond parameters between the phosphorylated peptide and the corresponding phosphorylation specific anti-human tau monoclonal antibody HPT-104, we perform dynamic force spectroscopy and compare the results to data using unsorted beads covered with the non-fluorescence peptide analogue. Finally, we demonstrate that the data variance of the relative binding frequency is significantly decreased by a factor of 3.4 using pre-sorted colloids with a homogeneous ligand coating compared to using unsorted colloids.
DOI