Background: In Western blotting, horseradish peroxidase (HRP) and alkaline phosphatase (AP) are commonly used as marker enzymes for color development. HRP is particularly popular due to its high specificity, small size, stability, and broad substrate compatibility. However, in certain cell types with a high mitochondrial content, endogenous peroxidase levels can be naturally elevated, leading to non-specific background signals or "false" bands on the membrane.
Method Improvement: To reduce the interference from endogenous peroxidase, we have optimized a simple yet effective pretreatment step. After preparing the gastric parietal cell lysate and mixing it with loading buffer, the sample was loaded onto an SDS-PAGE gel. Following electrophoresis and transfer to a membrane, the membrane was immediately incubated in 3% hydrogen peroxide (Hâ‚‚Oâ‚‚) for 15 minutes. This treatment effectively quenched the activity of endogenous peroxidase. Afterward, the membrane was washed thoroughly with TBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) and blocked with 5% skim milk before proceeding with standard Western blotting procedures.
Results: Before Hâ‚‚Oâ‚‚ treatment, a non-specific band at approximately 30 kDa was observed on the membrane. Even in the absence of primary or secondary antibodies, this band became visible during color development, indicating the presence of endogenous peroxidase activity. After the Hâ‚‚Oâ‚‚ incubation, the non-specific band disappeared entirely. This method proved highly effective for cell types with abundant mitochondria, such as hepatocytes or neutrophils, significantly improving the specificity and reliability of Western blot results.
Conclusion: By incorporating a brief Hâ‚‚Oâ‚‚ treatment after membrane transfer, researchers can minimize background noise caused by endogenous peroxidase. This simple modification enhances the accuracy of Western blot analysis, especially in samples where high levels of endogenous enzyme activity may interfere with the detection of target proteins. It's a practical and widely applicable technique that should be considered when working with tissues or cells known for their high mitochondrial content.
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