Loading controls – why bother?

Loading controls are essential for proper interpretation of western blots.

Loading controls can be used to normalize the levels of protein detected  by confirming that  protein loading is the same across the gel. The expression levels of the loading control should not vary between the different sample types.

Loading controls are absolutely essential to ensure the reliability of your data when comparing expression of a protein in different samples.

"To obtain any reliable information about the expression levels of proteins on western blots, it is essential that an appropriate loading control is used. This is an absolute requirement for publication-quality work." 

- Professor Tony Kouzarides, University of Cambridge

Why are loading controls essential?


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 comparisons are made of 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 allows the variation in binding to be corrected for.

Requested by referees

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 results obtained are valid.

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