Housekeeping Proteins as Internal Loading Controls

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

Housekeeping proteins such as tubulin, actin, and GAPDH are often used to normalize. In the past, researchers assumed that these proteins were constant in every cell type, because these proteins maintain basic cellular function. Housekeeping proteins are acceptable loading controls if expression is stable, but expression of these proteins can vary depending on your cellular context.

Housekeeping proteins won’t effectively normalize in every experiment, but that doesn’t mean they won’t work for any experiment. If you choose to use a housekeeping protein as your normalization strategy, be sure to validate it to confirm stable expression for your experimental context. As cell types, tissue types, disease states, and experimental treatments change, your internal loading control should remain constant.

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Here are some things to keep in mind:

  • Gene expression levels do not reliably predict protein abundance. Just because mRNA levels are constant, this does not mean protein levels will be similarly constant.
  • Biological factors, like tissue type, growth conditions, stage of development, and disease, may influence expression of housekeeping proteins. Without constant expression, housekeeping proteins are an unreliable way to normalize.
  • Housekeeping proteins are typically very abundant. The resulting strong bands freque[marketo-fat form=”1644″]ntly cause signal saturation, which reduces the accuracy of detection.

If you have validated that your housekeeping proteins are constant across all your experimental treatments, you can use them as a reliable loading control. Actin, tubulin, and COX IV primary antibodies can be used for two-color normalization.

Find out more about housekeeping proteins as internal loading controls in Western Blot Normalization: Challenges and Considerations for Quantitative Analysis.

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