Loading control antibodies

how to choose loading control for western blot

There are 3 things to consider when designing your WB with loading control.

1. Protein size: the loading control needs to have a sufficient size difference to your target protein. If the size are too close the bands will mush together data interpretation will be difficult. That said, the sizes should not be too different either.
2. Protein level: the loading control should have similar abundance to target protein. If too different, in the same development time, the more abundant protein will over develop while the less abundant protein will show weak or no signal.
3. Orthogonality: even though loading control proteins are typically ubiquitously and constitutively expressed, this is not absolute. Some conditions might affect these proteins still, and it is important to know and avoid using these affected proteins' antibodies as loading controls.