Atomic force microscopy identification of transcription factor NFκB bound to streptavidin–pin-holding DNA probe

Abstract

A novel method for identifying DNA-binding proteins from image analysis using AFM was developed. Here, transcription factor NFκB, which a well-studied example of transcription activator proteins, was used as a target protein. 5′-Biotinlynated double-stranded DNA probe was labeled site specifically through high affinity with streptavidin. When the biotinylated DNA fragments were incubated with the streptavidin at a 1:2 molar ratio of DNA:streptavidin, the overall efficiency of labeling was over 90%. The double-stranded DNA probes were immobilized on a mica surface by the adsorption of streptavidin that attached to the 5′-end of DNA and applied for selection of the target protein NFκB in solution and then AFM was used to image the DNA probe–NFκB complexes. The length of the distance between 5′-labeled streptavidin and NFκB bound on DNA probes from AFM images is 0.64, the normalized position of the NFκB binding site, and this result is in close agreement with the expected 299 and 167 bp values.