Western blots are packed with potential sources of variability. Variability that isn’t accounted for limits reproducibility and threatens your chances for publication-quality data. Normalization corrects for variability introduced during the process of Western blotting.
So what should you do to get more reproducible data? Use an internal loading control for each blot. Internal loading controls are endogenous sample proteins that are stably expressed and unaffected by experimental conditions.
Requirements for an Effective Internal Loading Control:
- Linear, proportional response. Signal intensity of the internal control should accurately reflect sample con¬centration and abundance of loading control over a wide range.
- Low biological variability. Your experimental treatments should not affect the expression of your internal loading control. For example, expression of some housekeeping proteins may vary in response to experimental conditions.
- Corrects for variation at all stages of immunoblotting. Your internal control should correct for variation that occurs throughout the Western blot process, including gel loading and transfer.
- Compatible with immunodetection. The strategy you choose shouldn’t interfere with effective down¬stream detection of your target proteins.
For more information about internal loading controls, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis