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Normalization with an Internal Reference Protein

What is an Internal Reference Protein?

Internal reference proteins are endogenous proteins found within your sample used for Western blot normalization. Internal reference proteins require secondary antibodies and unrelated proteins in addition to your target proteins.

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Housekeeping Proteins

Highly-expressed “housekeeping” proteins include actin, tubulin, and GAPDH. When using this strategy, it’s important to validate each protein for your experimental conditions, including each tissue, treatment, and cell density, to ensure that their expression doesn’t change.

“‘House-keeping’ proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.”

“Instructions for Authors.” The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 3 March 2016.

Jensen et al. (2013) PLoS ONE 8(4): e61952.

Housekeeping protein

Target protein

Fluorescent detection of internal loading control

Fluorescent detection of target protein

Signaling Proteins

In this two-color normalization technique, the target protein is used as its own internal control. A pan-antibody detects the unmodified total (pan) target protein, and a phospho-antibody detects only the phosphorylated proteins.

This strategy corrects for changes in overall level of the target protein, for more accurate phospho-analysis and reveals subtle mobility shifts caused by phosphorylation.

Controls are necessary to ensure that "epitope interference" does not occur (i.e., binding of one primary antibody that blocks binding of the other primary antibody to its epitope).

“Signals obtained using antibodies specific for phosphorylated epitopes should be normalized to the total protein level of the target protein.”

“Instructions for Authors.” The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 3 March 2016.

Mendonsa et al. ​PLoS ONE 4(2):e4655 (2009).

Target protein

Fluorescent detection of phospho-protein

Fluorescent detection of pan-protein


Normalize for Your Needs

When versatility is what you need, go with the normalization strategy that is best for you and your research. Regardless of your experimental conditions, Odyssey® imaging systems can provide accurate results based on any normalization strategy.

More about the Odyssey CLx Imaging System

“The brilliant signal-to-noise ratio in combination with the ability to truly quantify the data is really outstanding.”

Geir Bjørkøy, University College of Sør-Trøndelag

Total Protein Control

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