Quantitative Analysis of Protein-DNA Interaction by qDPI-ELISA

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1482; p. 49
• Main Authors: Fischer, Stefan M, Böser, Alexander, Hirsch, Jan P, Wanke, Dierk
• Format: Journal Article
• Language: English
• Published: United States 01-01-2016
• Subjects: Binding Sites; Chromatin - chemistry; Chromatin - genetics; Chromatin Immunoprecipitation - methods; DNA - genetics; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; Nucleotide Motifs - genetics; Protein Binding - genetics; Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry; Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics; Transcription Factors - chemistry; Transcription Factors - genetics; Quantitative analysis of DNA binding; Transcription factor-DNA binding kinetics; Cis-regulatory elements; Protein–DNA interaction; GFP-BPC6; Quantitative DNA–protein-Interaction-ELISA (qDPI-ELISA); Synthetic DNA probes; DNA binding
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-6396-6_4

The specific binding of DNA-binding proteins to their cognate DNA motifs is a crucial step for gene expression control and chromatin organization in vivo. The development of methods for the identification of in vivo binding regions by, e.g. chromatin immunoprecipitation (ChIP) or DNA adenine methyltransferase identification (Dam-ID) added an additional level of qualitative information for data mining in systems biology or applications in synthetic biology. In this respect, the in vivo techniques outpaced methods for thorough characterization of protein-DNA interaction and, especially, of the binding motifs at single base-pair resolution. The elucidation of DNA-binding capacities of proteins is frequently done with methods such as yeast one-hybrid, electrophoretic mobility shift assay (EMSA) or systematic evolution of ligands by exponential enrichment (SELEX) that provide only qualitative binding information and are not suited for automation or high-throughput screening of several DNA motifs. Here, we describe the quantitative DNA-protein-Interaction-ELISA (qDPI-ELISA) protocol, which makes use of fluorescent fusion proteins and, hence, is faster and easier to handle than the classical DPI-ELISA. Although every DPI-ELISA experiment delivers quantitative information, the qDPI-ELISA has an increased consistency, as it does not depend on immunological detection. We demonstrate the high comparability between probes and different protein extracts in qDPI-ELISA experiments.