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It is about the western blot question. The paper:http://www.sciencedirect.com/science/article/pii/S1931312813001145. In the western blot experiment in this paper, they use beta actin as control. And also I saw other research paper also use beta actin as control. So why is beta actin used, not other gene? is it because it expressed in all different kinds of cells?

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  • $\begingroup$ Other genes can be and are used. Basically any gene which is expressed constitutively at high levels and is a different size than your protein of interest can be used as a loading control. $\endgroup$ – canadianer Oct 31 '15 at 19:51
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β-actin belongs to a class of genes called housekeeping genes. Their proteins typically perform functions that are required across essentially all cell types (for example, β-actin is part of the cytoskeleton's microfilaments), and are supposed * to be not affected by experimental conditions. In Western blotting, they are run as a "loading control" to show that very similar amounts of protein were loaded in each lane. This is absolutely essential in some experimental systems where it may not be possible to always collect the exact same number of cells for each sample, such as when working with primary cells, tissue samples, or systems where you may be altering protein synthesis, cell size/number, or apoptosis.

There are many different loading controls to choose from, and some may be dictated by the cell/tissue type you are examining — for example, when working with stem cells, you may want to show that your experimental conditions didn't induce differentiation in the cells, so a pluripotency marker like Oct-4A or Sox2 may be chosen. In other cases, you may be examining fractionated lysates (a membrane preparation, just the nuclei, mitochondria, only the soluble fraction, etc.) and so may choose Na/K ATPase, Histone H3, COX IV, or GAPDH, respectively.

The choice of loading control can also be influenced by the size(s) of the protein(s) you are analyzing, as investigators frequently either strip Western blots and reprobe them with the loading control, or run two different species and detect with different colored secondary antibodies when performing fluorescent Westerns. In either case, you want your loading control to be a different size than your original targets.

Finally, all of this is assuming that the researcher has already attempted to load equal amounts of total protein per lane by using any one of a variety of methods for quantitating protein concentration. These include Bradford, Lowry, and BCA type assays, which may or may not be detergent compatible, staining the membrane after transfer with dyes such as Ponceau S, SYPRO Ruby, or India ink, or staining the gel with Coomassie or other proprietary Western-compatible stains.

*In reality, some scientists are becoming more and more aware that many experiments actually do affect a number of "housekeeping" genes, but when most see variations by, for example, Western blot or qPCR, they often choose a different loading control to use.

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  • $\begingroup$ Good answer. You might also add total protein as an option for a loading control, e.g. Comassie or Ponceau stains, or a Lowry/Bradford assay; this does not assume anything about a specific protein, but allows expressing the changes of protein X relative to total protein. $\endgroup$ – Roland Nov 1 '15 at 7:58
  • $\begingroup$ @Roland thanks for the comment. I had kind of assumed that the researcher would already have done a protein assay before loading the sample, but I can make that explicit. $\endgroup$ – MattDMo Nov 1 '15 at 16:53
  • $\begingroup$ I meant using total protein instead of housekeeping genes. (In my humble opinion the "housekeeping" gene concept is fundamentally flawed; typically such genes are in the end verified against total protein anyway, so why not use total protein in the first place.) $\endgroup$ – Roland Nov 1 '15 at 19:29
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Beta-actin control provides assurance that effect you see in protein expression is not due to overall protein synthesis change.

For example, if you study some illness, it might cause change in metabolism, weight loss, loss of ability to synthesize different proteins or absorb amino acids from diet. You have to control for that, and something as universal as actin is one option.

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