Visualization of single proteins from stripped native cell membranes: A protocol for high-resolution atomic force microscopy

Abstract

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.