在细菌中进行 FLAG 标记蛋白的 ChIP-qPCR。

ChIP-qPCR of FLAG-Tagged Proteins in Bacteria.

机构信息

Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

Centre for Microbial Cell Biology, Leiden University, Leiden, The Netherlands.

出版信息

Methods Mol Biol. 2024;2819:55-75. doi: 10.1007/978-1-0716-3930-6_4.

DOI:10.1007/978-1-0716-3930-6_4

PMID:39028502

Abstract

DNA-protein interactions occur in biological processes such as genome replication, gene transcription, DNA repair, and chromatin compaction and organization. Mapping the distribution of the DNA-bound proteins on the chromosome is essential for understanding their associated biological process. Chromatin immunoprecipitation (ChIP) involves the antibody-mediated enrichment of DNA fragments bound by a target protein and has become one of the most powerful techniques for exploring the distribution of proteins on the chromosome. By incorporating quantitative polymerase chain reaction (qPCR) downstream of the ChIP assay, ChIP-qPCR was developed to describe binding profiles of DNA-associated proteins at a candidate locus. In this chapter, we describe ChIP-qPCR. We provide a step-by-step protocol for the preparation of a ChIP library of a 3× FLAG-tagged protein in bacteria, describe how downstream qPCR experiments can be performed with the appropriate controls, and explain how the data is analyzed. This chapter provides reliable technical guidance for ChIP-qPCR studies in bacteria.

摘要

DNA-蛋白质相互作用发生在生物过程中，如基因组复制、基因转录、DNA 修复和染色质的紧缩和组织。绘制 DNA 结合蛋白在染色体上的分布图谱对于理解其相关的生物学过程至关重要。染色质免疫沉淀（ChIP）涉及抗体介导的靶蛋白结合的 DNA 片段的富集，已成为探索染色体上蛋白质分布的最有力技术之一。通过在 ChIP 测定下游结合定量聚合酶链反应（qPCR），开发了 ChIP-qPCR 来描述候选基因座处 DNA 相关蛋白的结合谱。在本章中，我们描述了 ChIP-qPCR。我们提供了一个逐步的方案，用于在细菌中制备 3×FLAG 标记蛋白的 ChIP 文库，描述了如何使用适当的对照进行下游 qPCR 实验，并解释了如何分析数据。本章为细菌中的 ChIP-qPCR 研究提供了可靠的技术指导。