title: FACS-sorted particles reduce the data variance in optical tweezers-assisted dynamic force spectroscopy measurements.
abstract: 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.
authors.affiliation: Department of Experimental Physics I, Leipzig University, Linnéstraße 5, D-04103 Leipzig, Germany. stangner@physik.uni-leipzig.de
authors.forename: T
authors.initials: T
authors.lastname: Stangner
authors.forename: D
authors.initials: D
authors.lastname: Singer
authors.forename: C
authors.initials: C
authors.lastname: Wagner
authors.forename: C
authors.initials: C
authors.lastname: Gutsche
authors.forename: O
authors.initials: O
authors.lastname: Ueberschär
authors.forename: R
authors.initials: R
authors.lastname: Hoffmann
authors.forename: F
authors.initials: F
authors.lastname: Kremer
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D008024:Ligands; D008301:Maleimides / Q000737:chemistry*; D008863:Microspheres; D052898:Optical Tweezers; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D016875:tau Proteins / Q000737:chemistry* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D008301:Maleimides; D010446:Peptide Fragments; D016875:tau Proteins; C043592:maleimide