New Protocols for Western Blot Normalization to Help You Get Published

Western blotting is the most widely used method for the detection and characterization of proteins. Although the basic elements of Western blotting remain unchanged, journal standards for publishing Western blots (e.g., JBC’s Instructions for Authors) have become more rigorous in recent years.

Are you interested in quantifying your proteins on your Western blot but are not sure how to manage Western blot variability and increase the accuracy of your results?

The key is to maximize Western blot accuracy and precision. This makes relative comparisons meaningful. How can you accomplish this? By reducing variability whenever possible with good experimental design. You can also correct for variability by using the appropriate internal loading controls for your Western blot normalization.

Normalization Protocols

LI-COR developed a series of protocols to help improve the quality of quantitative Western blots. Whether you are a beginner or a seasoned user, we can help you collect rock-solid data that will meet even the toughest publication standards.

The protocols cover key factors for success, data analysis and interpretation, and include links to additional educational resources for quantitative Western blotting.

Do you need help determining the linear range of your target protein and internal loading control, or validating your housekeeping protein, using REVERT total protein stain for normalization or using total and post-translationally modified proteins for normalization? If so, our tools, products, and services can help you get published.

These protocols are also available in an online format at

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