Loading controls
What is a loading control antibody?
Loading controls are proteins that exhibit a high but stable level of constitutive expression in the cell type or sample that you are studying. Housekeeping genes, such as actin, GAPDH, tubulin, or mitochondrial proteins, are usually suitable choices.
Loading controls confirm that protein loading is equal across each lane of a gel and, therefore, help to normalize the levels of your protein of interest. Loading controls also ensure the reliability of your data between samples, as the expression levels of the loading control should remain consistent across sample types.
Below is an example of negative and loading controls in a western blot (Figure 1).
Why should I use a loading control?
Quantification
When lanes have not been loaded evenly, loading controls can be used to quantify the protein amounts in each lane by using the density of the loading control band to correct for the differences in loading.
Equal transfer
Loading controls have a second role as a control in western blots. They can be used to check that there has been even transfer from the gel to the membrane across the whole gel. This is imperative when comparing the protein expression levels between samples.
Edge effect
This is an issue that is particularly important in signaling assays or experiments where a large number of lanes are being run at once. Proteins in the outer lanes of the gel are transferred to the membranes in a position close to the frame. This may result in more variation in binding compared to other areas of the gel. Loading controls can show if this effect has occurred and allow you to correct for the variation in binding.
Requested by referees
Using loading controls is essential for publication-quality work. As an example, to be published in many Nature journals, a paper must meet four general criteria, the first of which is that it must "provide strong evidence for its conclusions." This directly correlates to the necessity of controls to prove that the results obtained are valid.
How do I choose the right loading control?
Molecular weight compatibility
It is essential to choose a loading control with a different molecular weight than that of the protein of interest. This ensures that you can distinguish between the bands.
Stable expression across conditions
Choose a protein that is not affected by your treatment, cell type, or disease state. Common housekeeping proteins like β-actin, GAPDH, and tubulin are often used, but their expression can vary under stress, hypoxia, or differentiation
Detection method
Select a control that is compatible with your detection system (eg, HRP, fluorescent, or chemiluminescent).
Subcellular localization
Match the choice of control to the localization of your target, eg, cytoplasmic, nuclear, mitochondrial, etc.
Types of loading control
Use the table below to help select the right loading control to match your target localization.