Background: In Western blotting, horseradish peroxidase (HRP) and alkaline phosphatase (AP) are commonly used as marker enzymes for color development. Among these, HRP is widely preferred due to its high specificity, small molecular weight, stability, and broad substrate compatibility. However, in certain cell types that contain a large number of mitochondria, such as hepatocytes or neutrophils, the natural levels of endogenous peroxidase can be quite high. This may lead to the appearance of non-specific "fake" bands on the membrane, which can interfere with accurate interpretation of the results.
Method Improvement: To minimize the impact of endogenous peroxidase on the outcome of Western blot experiments, we implemented a modified procedure. After preparing the gastric parietal cell lysate and mixing it with the loading buffer, the sample was loaded onto an SDS-PAGE gel. Following electrophoresis, the proteins were transferred to a membrane. Immediately after transfer, the membrane was incubated in 3% hydrogen peroxide (Hâ‚‚Oâ‚‚) for 15 minutes to inactivate any endogenous peroxidase activity. After this treatment, the membrane was washed thoroughly with TBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) and then blocked with 5% skim milk powder before proceeding with standard Western blotting steps, including primary and secondary antibody incubation and detection.
Results: Before the Hâ‚‚Oâ‚‚ treatment, a non-specific band at approximately 30 kDa was observed on the membrane. Notably, even in the absence of primary or secondary antibodies, the presence of this band indicated the presence of endogenous peroxidase activity. After the Hâ‚‚Oâ‚‚ incubation, this background band was effectively eliminated. This method proves particularly effective for samples derived from cells with high mitochondrial content, significantly improving the specificity and reliability of Western blot results.
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