Atomic force microscopy identification of transcription factor NF kappa B 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 NFkappaB, 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 NFkappaB in solution and then AFM was used to image the DNA probe-NFkappaB complexes. The length of the distance between 5'-labeled streptavidin and NFkappaB bound on DNA probes from AFM images is 0.64, the normalized position of the NFkappaB binding site, and this result is in close agreement with the expected 299 and 167 bp values. (C) 2002 Elsevier Science (USA). All rights reserved.