Can the Same Loading Control Be Used for Two Proteins in Separate Western Blots?
In Western blotting, loading controls (e.g., GAPDH, β-actin) are commonly used for protein normalization. If two target proteins are presented in separate blots, the same loading control can be used for normalization, provided that the following conditions are met:
Same Sample Origin
Both the target proteins and the loading control must be derived from the same cell lysate or tissue extract.
Consistent Sample Processing
The two target proteins must undergo identical sample processing, including boiling time, electrophoresis conditions, and all other preparatory steps.
Uniform Transfer Efficiency
Protein transfer from the gel to the membrane must be consistent across all samples to avoid variations that could affect normalization.
Minimization of Experimental Artifacts
Experimental errors, such as inconsistencies in sample loading, electrophoresis duration, or transfer conditions, should be minimized to ensure reliable normalization.
Accurate Recording of the Loading Control Signal
The signal intensity of the loading control should be consistently recorded and applied in quantitative analysis.
Stable Expression of the Loading Control
The loading control must exhibit stable expression across different experimental conditions. If its expression is influenced by treatment conditions, it may not be a suitable reference for normalization.
When these conditions are met, using the same loading control for normalization remains valid, even if the two target proteins are displayed in separate blots. However, to ensure transparency and reproducibility, it is recommended to explicitly state in publications or reports that the same loading control was used and to clearly describe the experimental design and data processing methods.
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