Center for Reproductive Medicine, Affiliated Hospital 1 of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, PR China.
Department of Histology & Embryology, School of Basic Medical Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, PR China.
Anal Biochem. 2023 Jul 1;672:115179. doi: 10.1016/j.ab.2023.115179. Epub 2023 May 6.
The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.
Tris-EDTA 缓冲液的热诱导抗原修复技术的发展极大地提高了常规免疫组织化学检测中特定抗原的免疫染色（Krenacs 等人，2010）[1]。然而，关于 Tris-EDTA 缓冲液的热诱导抗原修复是否可以剥离 Western blot（WB）膜并允许连续重新探测，几乎没有证据。在这里，我们偶然发现，含有 0.01%吐温 20 的约 95°C Tris-EDTA 缓冲液可以反复剥离硝酸纤维素膜（NC）。电转移后，NC 印迹被浸泡在 Tris-EDTA 洗脱缓冲液中（约 95°C，10-25min），我们可以在连续化学发光检测中进行至少 5 轮洗脱（以下抗体用于：Vinculin、Atg7、Caspase-3、UBA5、JNK 和 ERK1/2）。在 WB 的重新探测过程中，NC 膜仍然显示清晰的 Western 信号和背景，而不会丢失转印的蛋白质。因此，本研究报告了 0.01%吐温 20 的热诱导抗原修复 Tris-EDTA 缓冲液的额外新作用。该方法更简单、更经济实惠，对硝酸纤维素纸也无害，这将有助于 Western blot 应用中的有效重新探测。
