High-throughput analysis of protein-DNA binding affinity.

摘要: 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 identify DNA motifs recognized a TF with lower affinity but retaining biological relevance. The use protein-binding microarrays (PBMs) offers high-throughput alternative for the identification specificities. PBM consists in an array pseudorandomized sequences optimized include all possible 10- 11-mer sequences, allowing determination binding eukaryotic TFs. PBMs can be synthesized several manufacturing companies single-stranded converted into double-stranded simple primer extension reaction. protein interest fused epitope tag then incubated onto PBM, specific DNA-protein complexes revealed series immunological reactions coupled fluorophore. After scanning quantifying PBMs, identified ready-to-use scripts, generating comprehensive accessible information specificity protein. This chapter describes detailed procedures preparation incubation recombinant protein, detection complexes. Finally, we outline some cues evaluating role obtained vitro.