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.

Full-length human cytomegalovirus terminase pUL89 adopts a two-domain structure specific for DNA packaging

“…Purified pUL89 or mutant proteins were purified as described above prior to solubilization in 4 x sample buffer (4% (v/v) ß-mercaptoethanol, 0.01% (w/v) bromophenol blue, 4% (w/v) glycerol, 4% (w/v) SDS, 0.2 M Tris-HCl (pH 6.8)), followed by separation on 10% (w/v) SDS-PAGE. Proteins were either stained with Silver/Coomassie or transferred to nitrocellulose sheets and subjected to immunoblot analysis as described previously [43]. The antibody mAbUL89 specific for pUL89 (1:1000; Fig 2A) was used as the primary antibody.…”

A simple and sensitive SYBR Gold-based assay to quantify DNA–protein interactions

“…Banasik and Sachadyn have demonstrated the feasibility of Pierce nickel-coated plates as a useful tool for testing DNA-binding affinity (Banasik and Sachadyn 2016). However, these assays are not easily scalable due to the limited protein binding capacity in these plates.…”