Types of Normalization Strategies

Normalization Webinar InvitationFor more information on Western blot normalization, watch these webinars:


Western Story - GlovedScientistsWhat normalization strategies are available to you when performing Western blots? The next four blog posts in this series will discuss the options that exist and considerations for use of each strategy.

An ideal internal loading control would have a linear, proportional response, be stably expressed in all experimental conditions, correct for variation throughout the whole process of immunoblotting, and be compatible with detection of your target proteins.

If you have the time to validate your loading control for each experiment, a housekeeping protein may work for you. You must validate all your housekeeping proteins to ensure stable expression.

If you’re studying protein modifications, like phosphorylation, ubiquitination, or glycosylation, then a multiplex normalization strategy with a signaling protein is recommended.

Total protein controls use all proteins present in the sample, and include total protein stains. If you don’t have time to validate, a total protein stain is best.

There are many ways to normalize. The best way depends on you and your experimental context. Watch for future blog posts about housekeeping proteins, signaling proteins, and total protein stains.

Find out more about normalization: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Understanding Western Blot Normalization

Normalization Webinar InvitationFor more information on Western blot normalization, watch these webinars:


chess piece - kingResearchers 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.

chess piece - bishopUnderstanding Western blot normalization will help you choose a strategy that fits the context and biology of your experiment.

This paper describes important considerations, strengths, and limits of commonly used normalization strategies:

Western Blot Normalization: Challenges and Considerations for Quantitative Analysis