Atomic force microscopy identification of transcription factor NFkappaB bound to streptavidin-pin-holding DNA probe.
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 167bp values.
Seong
Gi Hun
GH
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Yanagida
Yasuko
Y
Aizawa
Masuo
M
Kobatake
Eiry
E
D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D018625:Microscopy, Atomic Force / Q000379:methods; D016328:NF-kappa B / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry* / Q000648:ultrastructure
D015342:DNA Probes; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D001710:Biotin; D019809:Streptavidin