title: Detection and comparison of protein-DNA interactions using DNA-BIND plate and horseradish peroxidase-based colorimetric assay.
abstract: We describe a procedure for detection and comparison of protein-DNA interactions using DNA-BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA-BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein-DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins.
authors.affiliation: TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: Qin
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000906:Antibodies / Q000276:immunology; D003124:Colorimetry / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D050839:Interferon Regulatory Factor-7 / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding
chemical_list: D000906:Antibodies; D004268:DNA-Binding Proteins; D050839:Interferon Regulatory Factor-7; D009841:Oligonucleotides; D004247:DNA; D006735:Horseradish Peroxidase