For more information on Western blot normalization, watch these webinars:
- Western Blot Normalization: What You Need to Know
- Normalization Methods for Quantitative Western Blot Analysis
Researchers rely on Western blotting to detect target proteins in complex samples. This trusted technique is widely used to compare relative protein levels.
However, variability can creep into your Western blots through differences in sample preparation, sample loading, and transfer from gel to membrane. That’s why normalization is important. Normalization is the process of using internal loading controls to mathematically correct for sample-to-sample variation. These internal loading controls verify whether or not samples are uniformly loaded across the gel, confirm consistent transfer from gel to membrane, and enable comparison of relative protein levels between samples.
Normalization is meant to correct for small variation between samples, and can’t completely remove variability. If large data corrections are applied, accuracy may be affected. Normalization is a strategy to apply throughout your experiment, rather than a last step in the protocol. The more sources of variability you can reduce or eliminate, the more reproducible your experiment will be.
The role of an internal loading control is always to confirm the changes you see on the blot reflect actual change in the biology of your samples. To demonstrate statistically significant changes in the abundance of target protein, you need a reliable normalization strategy that fits the context and biology of your experiment. Effective, carefully-planned normalization will more accurately reflect the amount of protein in each lane.
This paper describes important considerations, strengths, and limits of commonly used normalization strategies: