Embedding and immobilizing protein molecules into two-dimensional protein arrays for single-molecule imaging by tapping mode atomic force microscopy

Soluble proteins, ferritin and catalase, were imaged as single molecules by tapping mode atomic force microscopy (AFM) in water. They were isolated and immobilized in two-dimensional (2D) arrays of smaller proteins: ferritin was embedded in 2D arrays of catalase, and catalase was embedded in streptavidin arrays. In the preparation of each sample, two kinds of proteins were dissolved simultaneously in a buffer solution, and were bound to a surface film of polypeptide spread at the air/buffer interface. The protein-bound film was transferred onto a hydrophobic surface of silicon wafer. The isolated molecules of ferritin and catalase protruded halfway from the surface of 2D arrays of majority constituents. The lateral dimensions of these molecules were of convolution with the cantilever tip with the end radius of approximately 3 nm. The characteristic shape or size of ferritin and catalase has been well captured as single molecules. This paper demonstrates that embedding protein molecules into protein arrays is a useful means for immobilizing proteins for single-molecule imaging by AFM.