A modified sensor chip for surface plasmon resonance enables a rapid determination of sequence specificity of DNA-binding proteins.
A novel method is described which rapidly determines specificity of DNA-binding proteins using a surface plasmon resonance (SPR) sensor chip. An oligohistidine-tagged DNA-binding domain of a transcription factor, NtERF2, was immobilised via nitrilotriacetic acid ligands to a sensor chip with an attenuated degree of carboxymethylation. DNA molecules were selected from a pool of randomised oligomers through binding to the immobilised protein and amplified by PCR. After several cycles of selection, during which binding was monitored by SPR, DNA sequences containing a consensus sequence were determined. The time necessary for one cycle is approximately 50 min, which is shorter than existing methods.
Hao
Dongyun
D
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Ohme-Takagi
Masaru
M
Yamasaki
Kazuhiko
K
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010940:Plant Proteins; D020349:Surface Plasmon Resonance / Q000379:methods*
D004268:DNA-Binding Proteins; D010940:Plant Proteins; C093680:ethylene-responsive element binding protein; D004247:DNA; D009571:Nitrilotriacetic Acid