Why is it important to validate housekeeping proteins before using them for Western blot normalization?
Housekeeping proteins (HKPs) are routinely used for Western blot normalization. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. However, expression of several HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression will introduce error and may alter data analysis and interpretation. In the instructions to authors for the Journal of Biological Chemistry, they state:
Before using a housekeeping protein for Western blot normalization, it is critical that you validate that its expression is constant across all samples and unaffected by your experimental context and conditions, especially if you have plans to publish.
As cell types, tissue types, disease states, and experimental treatments change, your internal loading control should remain constant.
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 frequently cause signal saturation, which reduces the accuracy of detection.
How do I validate housekeeping proteins?
The Housekeeping Protein Validation Protocol gives you step-by-step instructions on how to validate a housekeeping protein for use as an internal loading control. It also provides information on how to analyze the data in Image Studio™ software (download free Image Studio Lite) and how to quantify housekeeping proteins.
Detailed calculations and information on how to interpret the data will allow you to be confident in your validation process - and make the right decision for your Western blot normalization strategy.
When you have completed your housekeeping protein validation and have determined that the HKP expression is unaffected by your experimental conditions, you can use the Housekeeping Protein Normalization Protocol and proceed with using your validated HKPs for Western blot normalization and quantitative analysis. There you go!
Unlike what other vendors may be telling you, you CAN use housekeeping proteins for Western blot normalization - as long as you validate that their expression is not changing under your experimental conditions. Download your copies of Housekeeping Protein Validation Protocol and Housekeeping Protein Normalization Protocol and get started today.
LI-COR has several primary antibodies to housekeeping proteins that can be used for your normalization strategy, including actin, tubulin, COX IV, GAPDH, histone H3, and vinculin.
Other Protocols to Support Western Blot Normalization
LI-COR has several other protocols to help you meet new stringent publication guidelines and requirements. These are detailed protocols and include information on how to analyze and interpret your data.
- Determining the Linear Range for Quantitative Western Blot Detection
- Revert™ 700 Total Protein Stain Normalization protocol
- Pan/Phospho Analysis For Western Blot Normalization
With all of these protocols and our scientific experts, we can help you collect accurate, reliable Western blotting data. You will be confident in your results and your conclusions. When you submit your data for publication, you will be confident that you are meeting even the toughest publication standards. Protocols are also available in an online format at protocols.io
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