Normalization is often done by comparing the signal for the target protein to that of a “housekeeping protein.” A housekeeping protein is thought to be consistently expressed across different sample types. Its expression is believed to be unaffected by whatever intervention was carried out to produce the samples being compared.
Western blotting will tell you if your protein of interest is present in a sample. To determine if the amount of target protein is different between two or more samples, quantitative blotting is required. This involves:
- Ensuring measurements of the protein of interest are within the linear range of the detection system used, and
- Normalizing the measurements of the protein of interest to control for differences in the amount of sample loaded and/or any differences in transfer efficiency during the blotting protocol.
However, experimental conditions may change the expression of some commonly used housekeeping proteins. The expression level of the housekeeping protein must be similar to that of the protein of interest, otherwise one of the two proteins may not be within the linear range of detection. If the signal for the housekeeping protein is saturated under conditions needed to detect the target protein, then the housekeeping signal is not useful for normalization.