This article describes the use of loading controls, which are vital to the successful and correct interpretation of western blotting tests. Loading controls are required if western blotting results are to be interpreted correctly.
The use of loading controls helps to normalize the levels of protein that are detected in the sample, as they provide proof that the protein has been uniformly loaded across the gel. The levels at which the loading control is expressed should remain unchanged across the various types of sample.
Thus, it is crucial to use loading controls so that the data is known to be reliable when protein expression levels across a range of samples are being compared.
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, the University of Cambridge.
Why are Loading Controls Essential?
The lack of even loading across lanes can be compensated for by using loading controls to assess the amounts of protein added in each lane quantitatively. This is by employing the density of the loading control band to amend any variations in the loading.
Another important role of loading controls is their use as a control in western blots. They can be used to ensure the uniform gel-membrane transfer of the sample over the whole gel. This is vital when comparisons are made of the protein expression levels between samples.
When carrying out signaling experiments or any assay which involves running samples in many lanes simultaneously, the edge effect is very important. 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, a paper must fulfill four criteria overall if it is to be accepted for publication by many Nature journals. The first of these is the requirement that the paper, “provides strong evidence for its conclusions.” This directly correlates to the necessity of controls to prove that results obtained are valid.
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