Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK.
Methods Mol Biol. 2021;2263:369-379. doi: 10.1007/978-1-0716-1197-5_17.
The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein-DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.
蛋白质识别特定的 DNA 序列对 DNA 复制、转录和基因调控等基本生物过程至关重要。表面等离子体共振 (SPR) 技术非常适合测量这些相互作用，因为它是定量的、易于实现的、可重复的、可自动化的，并且只需要很少的样本。这通常涉及将生物素化的 DNA 直接捕获到链霉亲和素 (SA) 芯片上，然后让感兴趣的蛋白质流过并监测相互作用。然而，一旦 DNA 被固定在芯片上，就不能在不损坏芯片表面的情况下将其去除。此外，如果蛋白质-DNA 相互作用很强，那么在不损坏芯片表面的情况下将蛋白质从 DNA 上洗脱下来可能是不可能的。鉴于芯片成本高昂，这将限制可以测试的样本数量。因此，我们开发了一种可重复使用的 DNA 捕获技术，即 ReDCaT 芯片，它可以使单个链霉亲和素芯片重复使用多次，从而使该技术简单、快速且具有成本效益。我们将详细描述准备 ReDCaT 芯片、运行简单的结合实验以及分析数据的一般步骤。还将介绍一些其他应用。
