title: A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.
abstract: A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233, Gdańsk, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Banasik
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233, Gdańsk, Poland. psach@pg.gda.pl.
authors.forename: Paweł
authors.initials: P
authors.lastname: Sachadyn
mesh_terms: D001360:Avidin / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D003124:Colorimetry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D009532:Nickel / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D001360:Avidin; D006639:Histidine; D001710:Biotin; D009532:Nickel; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein
keywords: DNA mismatch; DNA–protein binding; His-tag; MutS; Nickel-coated microplate