Immuno-atomic force microscopy of purple membrane

The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of purple membrane while imaging in buffer solution. At forces greater than or equal to 0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic purple membrane surface was revealed. When the stylus was retracted, the scanned membrane area was relabeled with antibodies within 10 min, The extracellular surface of purple membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immune-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the purple membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.