Chapter title |
High-Throughput Analysis of Protein-DNA Binding Affinity
|
---|---|
Chapter number | 36 |
Book title |
Arabidopsis Protocols
|
Published in |
Methods in molecular biology, September 2013
|
DOI | 10.1007/978-1-62703-580-4_36 |
Pubmed ID | |
Book ISBNs |
978-1-62703-579-8, 978-1-62703-580-4
|
Authors |
Franco-Zorrilla JM, Solano R, José M. Franco-Zorrilla, Roberto Solano |
Editors |
Jose J. Sanchez-Serrano, Julio Salinas |
Abstract |
Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. |
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