Inactivation of horseradish peroxidase with crotonic acid for reprobing of western blotting

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. [Display omitted] •We developed a new type of stripping method for repeated detections of proteins on NC membranes.•20% crotonic acid an alternative to stripping buffer could strip and reprobe at least 3 times for NC blots.•The stripping method may save time and valuable samples.