Imaging the spatial orientation of subunits within membrane receptors by atomic force microscopy

Our experimental approach is based on the atomic force microscope (AFM) imaging of epitope-tagged subunits within membrane protein complexes purified in small amounts and decorated by anti-tag antibodies. Furthermore, we can produce simultaneous decoration of protein complexes using Fab fragments and IgG antibodies, which, combined with chemical modification of the substrate, allows us to determine the protein orientation across the cell membrane. Here, we describe a detailed protocol for membrane protein purification, AFM data collection, analysis, and interpretation of results. The protocol also covers basic AFM instrument settings and best practices for both observation of membrane protein complexes by AFM and automatic detection of the structures by an in-house algorithm. Once a sufficient number of membrane protein complexes have been visualized by AFM, data acquisition and processing can be completed in approximately 10 min using a scanning surface of 1 μm(2).