Loading control antibodies are a critical Western blotting tool that is often overlooked. They play an important role, ensuring that that you have loaded and transferred equivalent amounts of protein across your Western blot. If you are planning on comparing the protein level across samples, use a control to normalize your data for proper interpretation. This is especially important when quantitative data is required as you will need to compensate for uneven sample loading or transfer. Also, if you are planning on publishing your data, a loading control is now essential due to increased scrutiny of data.
The controls may be used simultaneously with your target antibody or sequentially after you have stripped the blot. However, simply using a loading control is not enough. It is important to choose the right one for your experiment. This means that you will need to take the following into consideration when making your selection:
- The molecular weight of your target protein should be differentiated from the control.
- Choose a loading control with a high level, constitutive expression in your sample.
- Signal of loading control must be in the same linear range as your protein of interest.
Use Table I below to select the best loading control for your experiment.
It is also a good idea to double check that your experimental conditions do not impact the loading control that you have selected. The most commonly used loading controls are Beta Actin, Beta Tubulin and GAPDH. Depending on the cells or tissues that you are working with the expression level of these controls may vary, making quantitation inaccurate. Chemical, pharmacological treatments and alterations in environment (i.e. hypoxia) may also interfere with the expression level of the control proteins. Before choosing your loading control ask yourself a few questions…
- Is the comparison between different treatments within the same cell line/tissue?
- Is the comparison between different cell lines/tissues?
- Is the comparison between normal cells/tissues to diseased samples (i.e. cancer)?
If you answered yes to any of the above questions, extra care should be taken to select a loading control that will not be impacted. It is also advised that you run a quick experiment to confirm that your selected control is appropriate.
Once you have selected the best control for your experiment enjoy greater confidence in data that you can trust! Try our products with enhanced sensitivity and low background to get the highest quality data for your next publication.