Imaging and Force Spectroscopy of Single Transmembrane Proteins with the Atomic Force Microscope

The atomic force microscope (AFM) has opened avenues and provided opportunities to investigate biological soft matter and processes ranging from nanometer (nm) to millimeter (mm). The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever-aptly termed as a "lab on a tip"-can be used as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples have convincingly established AFM as a tool to study the mechanical properties and monitor processes of single proteins and cells with high sensitivity, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of transmembrane proteins in a lipid bilayer (reconstituted or native). Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theory and practical skills.