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Normalization is Critical

for Quantitative Western Blot Data

How Do Your Western Blots Measure Up?

Publishers and funding agencies are redefining “publication-quality” data, which may change how you capture, analyze, and present quantitative Western blot data. If you want rock solid data that nobody can question, it’s critical to understand Western blot normalization.

Many strategies for Western blot normalization exist – like total protein stains, housekeeping proteins, Stain-Free™ detection, and signaling proteins. The strategy you choose depends on the context, biology, and goal of your experiment.

Accurate data measurement and analysis is the foundation of your research findings. Image file modifications using unsupported image editing and analysis software programs can compromise the integrity of your data. Ensure that you are using a software program designed to analyze the results of your Western blot experiments that is compatible with your detection system. See a list of other best practices for imaging and publishing Western blot data.

“Oddly, the most pervasive challenges to published data we see at Cell relate to loading controls. There seems to be some misalignment among scientists regarding the importance and meaning of the actin bands in a standard western blot.”

Marcus, E. Credibility and Reproducibility. Cell. 159(5): 965-966 (2014).

What’s In a Number?

Typically, the goal of quantitative Western blotting is to determine relative changes between different samples. The immunoblotting process itself introduces variability that you may not always be aware of – or even be able to prevent entirely.

The New Normal

In a perfect world, your data would directly relate to the biological composition of your samples. Sadly, real data are messy and complicated, because experiments are messy and complicated. It’s important to reduce variability wherever you can, but quantitative numbers are meaningless if they haven’t been normalized.

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What Is Normalization?

In general terms, normalization compares target protein signal intensity (unknown) to an internal loading control (known), to generate accurate and reproducible quantitative Western blot data.

Internal loading controls are proteins present within your sample.

Normalization uses these internal loading controls to mathematically correct for unavoidable sample-to-sample and lane-to-lane variation.

Which Strategy is Best?

There are many ways to normalize, and no single normalization strategy is always the best choice. Choose a strategy that fits the context, biology, and goal of your experiment.

Total Protein Control

Use all proteins present in the sample to normalize.

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Internal Reference Protein

Use a single endogenous protein to normalize, e.g. a “housekeeping” protein.

Learn More
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