title: Reflection interference contrast microscopy combined with scanning force microscopy verifies the nature of protein-ligand interaction force measurements.
abstract: The integration of a stand-alone scanning force microscope (SFM) scanner with a reflection interference contrast microscope (RICM) makes it possible to measure directly the separation distance between the SFM probe and the sample surface. The SFM-RICM combination, when applied to the force measurements between ligand-derivatized SFM probe and a protein receptor-derivatized surface, showed that the anomalous force discontinuities often observed for such interacting pairs were indeed a real behavior characteristic of a particular experimental configuration. Apart from small discrepancies due to transient damping, commercially available cantilevers did behave in an ideal mechanical fashion, thus indicating that protein-ligand unbinding events were occurring at distances much larger than their maximum extended length. This external verification of separation distance requires a closer examination of the physical events occurring upon detachment of the surfaces. An alternative interpretation of such force measurements is proposed here in which the protein and/or ligand immobilization chemistry is called into question.
authors.affiliation: Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112 USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Stuart
authors.forename: V
authors.initials: V
authors.lastname: Hlady
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D008857:Microscopy, Interference / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008024:Ligands; D011506:Proteins