What Makes Loading Controls Important?

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?

Quantification

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.

Equal Transfer

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.

Edge Effect

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.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Jun 6, 2019 at 12:37 PM

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