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
What 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