How to choose the right loading control for your experiments
Loading controls are essential in western blot experiments that ensure an accurate comparison between test samples. Typical examples of good loading controls are proteins that exhibit high-level, constitutive expression in the cell type or sample you are studying or are markers of a particular cell compartment.
Loading controls provide a consistent baseline for comparing samples. They are critical to the reliable interpretation of western blot data and the publication of that data in peer-reviewed journals. Even if you have used a biochemical assay (e.g. Bradford, BCA, etc.) to measure the total protein content of your sample in order to load equivalent amounts of protein in each well, it is still imperative to use a loading control to confirm.
Loading controls are also essential to confirm the quality of your samples and experimental set-up – if the expression of a loading control is not as expected, this could indicate a number of issues including
· problems with sample preparation,
· improper loading,
· incomplete protein transfer from gel to membrane, or
· edge effect (when the lanes at the outer edge of the gel show incomplete transfer to the membrane).
There are many factors to consider when choosing a loading control.
Sample type
The choice of the correct loading control depends on the sample type you are analyzing and the cell manipulations you have performed as part of your experimental design. For example, when investigating different conditions (e.g.siRNA KD v contol) of your protein of interest in whole cell lysates, you should choose a loading control that is expected to be present in consistent amounts in each sample– a popular choice is GAPDH, a common cytoplasmic housekeeping protein with ubiquitous tissue distribution. However, if you are performing subcellular fractionation of your sample to investigate the subcellular location of your target protein, then loading controls specific to your fractions are most suitable, eg, cytoplasmic-GAPDH, nuclear-LaminB, chromatin-histone H3.
Molecular weight
Another key consideration is that the molecular weight of the loading control should be different from that of your protein of interest so that they can be easily distinguished on the gel. As such, it’s important to have a range of proteins to choose from for any loading control.
Range of detection
Ideally, a loading control should express at a similar level across a range of sample concentrations to ensure the intensity of the loading control and protein of interest fall within the linear range of detection. If your target of interest is a low-abundance protein, this should be taken into account when choosing a loading control. In this case, a higher than-average amount of total protein may be required per well in order to visualize a low-abundant protein. If the higher amount of protein causes the loading control to fall outside the linear range of detection or the sample exceeds the transfer capacity of the membrane, this can impact the correct interpretation of the results. This can give the appearance of equal loading whilst masking small differences. Additionally, this can make exposure of the membrane to the film problematic.
Experimental conditions
Another factor to consider when selecting a loading control is the experimental manipulation used to prepare your samples. There are situations where the expression of even commonly used loading controls can be altered; for example, GAPDH levels can be altered in hypoxic conditions, alpha-tubulin can be altered in anti-cancer or anti-fungal treatments, and beta-actin levels can be affected by the cellular stress response.