For more information on Western blot normalization, watch these webinars:
- Western Blot Normalization: What You Need to Know
- Normalization Methods for Quantitative Western Blot Analysis
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.
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.