School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 32500, PR China.
School of 2nd Clinical Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, PR China.
Anal Biochem. 2023 Mar 15;665:115070. doi: 10.1016/j.ab.2023.115070. Epub 2023 Feb 1.
Inactivation of horseradish peroxidase (HRP) treatment is a conventional preference to stripping for sequential detections of different proteins of chemiluminescent western blotting (WB). However, little evidence exists on whether other chemical substances treatment can affects the biological activity of HRP during stripping and re-probing of WB blots. Here, we successfully develop 20% crotonic acid (CA) as an alternative to stripping to inhibit HRP used for sequential chemiluminescent WB on polyvinylidene difluoride (PVDF) and Nitrocellulose (NC) membrane. Moreover, NC blots incubation in CA (40 °C, 30min) allow us to perform three round HRP inhibition in sequential detections without losing transferred proteins and damaging membrane. Hence, the method will help us save time and valuable samples without the need to rerun gels.
辣根过氧化物酶（HRP）失活处理是化学发光免疫印迹（WB）连续检测不同蛋白质时常用的脱膜方法。然而，关于其他化学物质处理是否会影响 WB 膜脱膜和再探测过程中 HRP 的生物学活性，目前证据有限。本研究成功开发了 20%巴豆酸（CA）作为脱膜替代物，用于抑制 PVDF 和硝酸纤维素（NC）膜上连续化学发光 WB 中的 HRP。此外，NC 膜在 CA（40°C，30min）孵育可在连续检测中进行三轮 HRP 抑制，而不会丢失转印蛋白和损坏膜。因此，该方法有助于我们节省时间和宝贵的样本，无需重新跑胶。
