Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot

Chemiluminescent western blot (WB) is often performed sequentially for detection of overlapping proteins; in between, prior antibodies must be stripped or the conjugated horseradish peroxidase (HRP) inactivated. However, often, stripping either is insufficient to remove all the bound antibodies or causes protein loss, whereas treatment with hydrogen peroxide, a popular way to inactivate HRP, may affect epitope recognition as the authors previously reported. To date, an ideal method for sequential chemiluminescent WB is still missing. Here it is demonstrated that acid equivalent to 10% acetic acid can efficiently inactivate HRP, allowing sequential probing without protein loss or epitope damage.