How to Choose a Loading Control for Western Blots

Loading controls are needed to interpret western blots. The following guide explains what they are, and how to choose the right one. You can see a full list of loading controls available from Hello Bio here.

What are loading controls?

Loading controls are antibodies against a different target to the protein of interest used in immunoblotting (western blotting). These targets are often highly expressed housekeeping proteins who’s expression is stable. Loading controls are essential when the relative expression of proteins is being compared in a gel and are used to:

- Ensure that the loading of proteins is uniform across the whole gel. Where there has been unequal loading, the loading control can be used to account for this.

- Ensure that there has been equal transfer of proteins from gel to membrane across the gel

 

Choosing a loading control

There are several key principles that need to be followed when choosing a loading control for immunoblotting:

- The loading control and target of interest should have different molecular weights to ensure they do not overlap on the gel

- The loading control expression should not be affected by any experimental manipulation between samples.

- The loading control should have high levels of expression in the sample

- The loading control should be in the portion of the gel where there is linear separation otherwise it will not be possible to quantify it.

 

Use the table below to select the right loading control for your sample type. Remember to select a loading control that has a different molecular weight to the protein of interest. This ensures that you will be able to distinguish between the bands.