Loading controls serve as a vital method of verifying the results acquired through western blotting by normalizing the protein levels detected by confirming that protein loading is the same across the gel.
When western blotting, there can be numerous roadblocks to reliable results. Nearly every step of the experiment, from sample preparation to the detection methods, can pose a problem. When normalizing the protein levels detected, loading controls serve as an indispensable method of verifying the results. Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample. They are essential for several reasons:
Edge Effect:
When multiple lanes are run on a blot, the outer lanes may experience the "edge effect.” The edge effect occurs when proteins drift towards the center; luckily, this can be removed through the use of a loading control, fine-tuning the entire blotting process by correcting the variation in binding. Loading controls can be further used to minimize the impact of varying protein expression levels.
Equal transfer:
Loading controls are 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.
Quantification:
Most importantly, when lanes have not been loaded evenly, loading controls can quantify the protein amounts. The density of the loading control band is used to correct for the differences in loading.
How to choose a loading control:
The proteins serving as loading controls must fit specific criteria.
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The protein levels of loading controls must remain constant (relative to the total protein content) under the test conditions (such as across treatments or developmental stages).
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The detection bands of loading controls must not interfere with those of the protein(s) of interest (i.e., they should have substantially different molecular weights).
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The detection limits for the loading controls and the proteins of interest are within dynamic ranges.