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A fundamental prerequisite for the investigation of biological specimen by force microscopy is their immobilization to inert surfaces. In this review, we report recent anchoring protocols and their application for atomic force microscopy single molecule recognition studies of our group. The surface chemistry by which ligands are covalently coupled to force microscopy tips is described in detail. Poly(ethylene glycol) was employed as a spacer for the ligands, being a most suitable crosslinker for force spectroscopy and microscopy experiments. Furthermore, different strategies for the anchoring of isolated proteins, lipids, vesicles, and cells, containing receptor binding epitopes, to probe surfaces are discussed. The basic principles of force spectroscopy are outlined and the successful application of this technique to several biological systems is demonstrated. It is shown that force spectroscopy reveals information about kinetic rates, affinities, and the dynamic structure of the binding pocket. This technique shows great potential for investigating the molecular dynamics of ligand–receptor binding and the epitope mapping of receptors. |
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