In vitro Protein-DNA Binding Assay (AlphaScreen® Technology)

Mika Nomoto; Yasuomi Tada; Hironaka Tsukagoshi

doi:10.21769/BioProtoc.3155

In vitro Protein-DNA Binding Assay (AlphaScreen^® Technology)

Bio Protoc. 2019 Feb 5;9(3):e3155. doi: 10.21769/BioProtoc.3155.

Authors

Mika Nomoto^{1

2}, Yasuomi Tada^{1

2}, Hironaka Tsukagoshi³

Affiliations

¹ Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
² Center for Gene Research, Nagoya University, Nagoya, Japan.
³ Faculty of Agriculture, Meijo University, Nagoya, Japan.

Abstract

Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our in vitro transcription/translation system and AlphaScreen^® technology. The in vitro transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen^® system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions in vitro. Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.

Keywords: AlphaScreen; DNA binding; FLAG-tag; In vitro transcription/translation; Protein-DNA interaction; Transcription factor.