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
Besides housekeeping proteins and total protein controls, signaling proteins are another option for normalization. This approach is particularly useful for relative analysis of post-translational modifications such as phosphorylation. The method combines two primary antibodies raised in different hosts: a phospho-specific antibody (or other modification-specific antibody) and a pan-specific antibody that recognizes the target protein regardless of its modification state. Fluorescently-labeled secondary antibodies are used to simultaneously detect and discriminate the two signals with digital imaging. Phospho-signal is then normalized against the total level of target protein, using the target protein as its own internal control.
This is a great strategy to use if you’re studying protein modifications. Bakkenist et al. examined the possibility of binding interference from combined phospho-specific and pan antibodies, but detected little or no effect.
Advantages of Phospho-Analysis with Signaling Proteins:
- You can detect both unmodified and modified forms of your target protein on the same blot, in the same lane.
- No error is introduced by stripping and reprobing. Stripping and reprobing of blots can introduce detection artifacts and cause loss of blotted proteins from the membrane.
- Accuracy is improved by correcting for loading and sampling error
Find out more about multiplex analysis using signaling proteins: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis