Western bolting is a well-established method that is currently relied on in biology laboratories for determining the specific proteins and their abundance within a given sample of cells or tissue.
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Loading controls are an essential part of the Western blot process, and they are necessary for accurate interpretations to be made.
Loading control is a protein, usually one that exhibits high-level constitutive expression within the sample or cell type under investigation. Proteins such as beta-actin or GADPH are those with high and ubiquitous expressions that are commonly used as loading controls.
These proteins are used to indicate the equal loading of samples across all wells. They also express the proper transfer of proteins onto the membrane during the western blotting process.
Overall, these proteins are a requirement to be able to accurately determine protein expression in Western blotting and to precisely read results.
Why are loading controls necessary for Western blotting?
Loading controls serve numerous purposes in a Western blot investigation. They are essentially used to normalize the levels of protein detected within a sample by ensuring that protein loading is the same across the gel. Below, the four main reasons why loading controls are crucial are outlined.
Firstly, loading controls provide a method of quantification, enabling researchers to calculate the protein amounts in each lane when they have not been loaded evenly. To do this, the density of the loading control band is used to correct for the differences in loading.
An equal transfer is the second application of loading controls in Western blots. It is vital that when comparisons are made between the protein expression of different samples, each sample has had an even transfer from the gel to the membrane across the whole gel. Loading controls are used to check these transfers.
The interpretation of Western blot results faces a particular challenge when a large number of lanes are being run simultaneously. Because proteins within the gel’s outer lanes get transferred into the membranes positioned close to the frame, this can result in the edge effect, where there is more variation in binding at the edges in comparison to the rest of the gel. Loading controls can signify if this has occurred and can allow researchers to correct for it.
Finally, most research projects seek to be published in renowned journals. These journals require research to meet certain criteria, such as that researchers must provide strong evidence for their conclusions. Controls included within research methodologies help present a case for strong evidence.
Advantages of loading controls
Failing to include loading controls when carrying out Western blot techniques can lead to research not being published because it is not verified. Therefore, an advantage of including loading controls is that the research meets the criteria for being published.
Also, loading controls add to the level of quality of the data collected as it can be beneficial in correcting for errors and ensuring the accuracy of the information used to make vital conclusions. Loading controls limit inconclusive or incorrect results, and therefore save research projects time and money in re-running procedures.
Overall, loading controls enhance the reliability and efficiency of the Western blot method and enable studies using them to meet the criteria for being published.
Types of loading controls
Currently, numerous proteins are used as loading controls. The type of protein chosen for use in a study will often depend on what is being investigated and what is sought to be found out from the sample. The most popular controls are:
Whole-cell and cytoplasmic extract applications are perfect for Actin use. However, because it is a component of muscle action, it cannot be used in skeletal muscle samples. Actin exists in many forms, making it incredibly versatile.
Beta-Actin (42 kDa)
One popular form of Actin is Beta-Actin (42 kDa). It can be used in samples of all eukaryotic cell types, which is why it is a popular choice of loading sample. Additionally, its expression levels do not significantly vary due to cellular treatment, again making it a widely suitable control.
Glyceraldehyde 3-phosphate dehydrogenase, known as GAPDH, is a whole-cell and cytoplasmic extract loading control. It also acts as a catalyst for the breakdown of glucose and carbon molecules. Its expression level does vary between tissues, which is an important factor to bear in mind when considering it as a loading control.
Porin is most commonly used in samples of mitochondrial proteins. It is a voltage-dependent anion-selective channel protein that is a component of numerous tissue types.
Lamin B1 is considered to play a role in nuclear stability within a cell. It is suitable as a loading control for nuclear protein envelopes, but it should only be used if the nuclear envelope is available.
The TATA-binding protein, known as TBP, is a transcription binding protein that works with RNA polymerase II. It is another common loading control for nuclear proteins.
To accurately measure protein expression and read Western blot results, the use of loading controls is essential. These proteins are used to augment the overall reliability and efficiency of the Western blot method and allows studies to meet the criteria for publication. While numerous loading controls exist, some of the most common include Actin (particularly the Beta-Actin form), GAPDH, Porin, Lamin B1 and TBP.
• Eaton, S., Roche, S., Llavero Hurtado, M., Oldknow, K., Farquharson, C., Gillingwater, T. and Wishart, T. (2013). Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting. PLoS ONE, 8(8), p.e72457. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072457
• Ghosh, R., Gilda, J. and Gomes, A. (2014). The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review of Proteomics, 11(5), pp.549-560. https://www.tandfonline.com/doi/abs/10.1586/14789450.2014.939635
• Murphy, R. and Lamb, G. (2013). Important considerations for protein analyses using antibody-based techniques: down-sizing Western blotting up-sizes outcomes. The Journal of Physiology, 591(23), pp.5823-5831. https://physoc.onlinelibrary.wiley.com/doi/full/10.1113/jphysiol.2013.263251