Higashi Sayuri L, Yagyu Kazuya, Nagase Haruna, Pearson Craig S, Geller Herbert M, Katagiri Yasuhiro
Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University.
Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health.
J Vis Exp. 2020 Sep 26(163). doi: 10.3791/61657.
A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.
蛋白质免疫印迹法（也称为免疫印迹）是生物医学研究的经典方法。它通常用于确定特定蛋白质的相对大小和丰度以及蛋白质翻译后修饰。这项技术历史悠久，因其操作简单而仍被广泛使用。然而，蛋白质免疫印迹过程众所周知需要数小时甚至数天才能完成，一个关键瓶颈是长时间的孵育时间限制了其通量。由于抗体从大量溶液扩散到膜上固定的抗原的速度很慢，所以需要这些孵育步骤：膜附近的抗体浓度远低于大量溶液中的浓度。在此，我们提出了一项创新，通过循环排出和补充（CDR）抗体溶液来改善抗原结合，从而显著缩短这些孵育时间间隔。我们还利用了一种免疫反应增强技术来保持检测的灵敏度。CDR方法与市售免疫反应增强剂相结合提高了输出信号，并大幅缩短了抗体孵育时间。由此产生的超高速蛋白质免疫印迹法可在20分钟内完成，且灵敏度没有任何损失。该方法可应用于使用化学发光和荧光检测的蛋白质免疫印迹。这个简单的方案使研究人员能够更好地探索许多样品中蛋白质表达的分析。
