Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Korea.
KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Korea.
Electrophoresis. 2018 Dec;39(23):2974-2978. doi: 10.1002/elps.201800195. Epub 2018 Sep 2.
Western blot is a principal technique for the detection of specific proteins in various biology disciplines. The antibody incubation is an imperative step in western blot. Antibody incubation by mild shaking on a rocker is generally used to facilitate mixing of antibodies. However, mild shaking is an inefficient process to remove antibody depletion layer on blotting membrane and requires hours of incubation time to achieve antibody binding to target proteins. We propose an alternative method of cyclic draining and replenishing (CDR) incubation of antibody solution using a rotational incubation chamber. The study demonstrated that rotational CDR incubation could shorten antibody incubation time with enhanced sensitivity. Moreover, rotational CDR incubation could achieve a stronger antibody binding with lower antibody concentration when compared with batch incubation. In addition, rotational CDR incubation significantly improved the detection of low abundance proteins. This simple modification in western blot procedure makes it rapid, sensitive, and cost-efficient.
蛋白质印迹法是各种生物学学科中检测特定蛋白质的主要技术。抗体孵育是蛋白质印迹法中的一个必要步骤。在摇床上进行温和的晃动通常用于促进抗体的混合。然而，温和的晃动是一种低效的过程，无法去除印迹膜上的抗体耗尽层，需要数小时的孵育时间才能实现抗体与靶蛋白的结合。我们提出了一种使用旋转孵育室进行循环排干和补充（CDR）孵育抗体溶液的替代方法。研究表明，旋转 CDR 孵育可以缩短抗体孵育时间，提高灵敏度。此外，与批量孵育相比，旋转 CDR 孵育可以用更低的抗体浓度实现更强的抗体结合。此外，旋转 CDR 孵育显著提高了低丰度蛋白质的检测能力。这种对蛋白质印迹法的简单改进使其快速、灵敏且具有成本效益。
