This is a review of beta-actin antibodies (mainly as an internal loading control for Western blot), based on formal publications that Labome has surveyed systematically. Labome is continuously curating antibodies cited in formal publications and an updated enumeration of beta-actin antibodies in Labome's Validated Antibody Database (VAD) is here. There are numerous publications using beta-actin antibodies; our systematic and random sampling ensures that our results and conclusions are representative (see Table 1). This review aims to facilitate Labome visitors in locating the best-suited beta-actin antibodies. Beta-actin antibodies from different suppliers are linked below or can be searched here. The article also addresses some of the commonly asked questions about beta-actin antibodies.

Beta-actin is one of the most commonly used loading controls for Western blot, in addition to tubulin and GAPDH.

Figure 1. A typical application of beta-actin antibody as the Western blot loading control, from [1].

The actin family of proteins are involved in muscle contraction and cytoskeleton structure. Beta and gamma forms are the only two non-muscle members. They play important rules in basic cellular processes, and are expressed in all cell types. Their sequences are highly conserved across species. Beta-actin proteins are identical in human, mouse, and chicken.

Beta-actin is the most commonly used loading control for western blot (see Labome's dedicated discussion on loading control for western blot). Table 1 lists the major providers of monoclonal anti-beta-actin antibodies among the publications Labome has surveyed. Polyclonal anti-actin antibodies are also used, for example, anti-β-actin pAb-HRP-DirecT (PD030) from MBL [15].

The most cited anti-beta-actin antibody clone is AC-15, which was used in Western blot, for example, in order to normalize Western blot signals for quantitative measurements [3] and intein-mediated protein trans-splicing [11]. Its specificity has been confirmed in knockout studies [16, 17]. Other commonly used clones include AC-74 (MilliporeSigma A5316 [18] and A2228 [19] ) and C4 (SCBT sc-47778 [20] ; MilliporeSigma MAB1501R) [21].

AC-15 clone was characterized and reported by Dr. J. Victor Small's lab at the Institute for Molecular Biology, Austrian Academy of Sciences in their 1994 publications [22, 23]. AC-15 belongs to the subclass IgG1, and was raised in mice against the N-terminal synthetic peptide of beta-actin, with the sequence of AcD-D-D-I-A-A-L-V-I-D-N-G-S-G-K coupled to keyhole limpet hemocyanin. The clone specifically targets the beta isoform, with no cross-reactivity against the other 5 isoforms of actin family. This clone is available from most antibody suppliers.

Dr. Mario Gimona, then a Ph.D. student in Dr. Small lab has kindly provided the following account of history about the clone AC-15 to this article.

While conducting experiments in 1993 at the Weizmann Institute of Science in Rehovot, Israel, I was invited to visit the facilities of BIOMAKOR, a company focusing on the development of monoclonal antibodies. Dr. Harry Langbeheim and Dr. Zeev Lando of BIOMAKOR instructed me on their latest developments that included isoform-specific actin antibodies (at that time excellent probes for muscle actins and pan-actin Abs were available, but a beta-isoform-specific probe was lacking). I was given the chance to take some of the putative beta-actin specific probes (AC 15 and AC 74) back to Salzburg to test the Ab in our then lab in Salzburg.

With the help of Prof. Small and Dr. Alison North in Salzburg, and together with our collaborator, the actin isoform expert Prof. Joel Vandekerckhove from the University of Ghent, Belgium, we characterized the Ab by light and electron microscopy, as well as 1D and 2D Western Blotting (on whole cell lysates and on isoform-specific peptides).

The probe demonstrated excellent specificity and worked well in all techniques and fixation methods used.

Dr. Langebheim, Dr. Lando and BIOMAKOR generously granted us the publication of 2 initial papers in 1994 and provided us with a generous supply of the AC-15 Ab for many years.

To my knowledge the company BIOMAKOR later on went to become part of SIGMA.

Due to the prevalence of anti-actin antibodies in Western blot as a loading control and the prevalence of Western blot analysis in the overall body of literature, this beta-actin clone is likely the most cited antibody clone among all antibody clones in the literature.

It must be noted that beta-actin is not an appropriate Western blot loading control in some circumstances. At a higher total protein loading level, beta-actin specific antibodies may not be able to detect the differences in protein loading levels [24]. This is likely due to that beta-actin is one of the dominant cellular proteins. This problem may not be alleviated by diluting anybody working solution or shortening the incubation time [24].

In addition, the beta-actin expression might change during growth and differentiation, or in disease states [25], or in different tissues and in different portions of the same sample tissues [26]. It is important to evaluate the consistency of beta-actin expression over, for example, the total protein, among different samples.

Some of the commonly asked questions about beta-actin antibody are addressed below.

There are a number of reasons. The first one to check is whether the anti-beta-actin antibody is effective. One way to accomplish that is to reduce the dilution of the anti-beta-actin antibody, and check to see whether there is still any band at all, and/or try to use anti-beta-actin antibodies from high-quality suppliers. The second thing to check is whether the anti-beta-actin anybody is suitable for Western blot; some of the anti-actin antibodies are good for immunocytochemistry or immunohistochemistry, but not for Western blot. The third thing to check is whether the proteins are transferred evenly from the gel to the membrane. This can be checked through Ponceau S staining. The fourth thing to check is whether beta-actin is expressed in the sample tissue or cells; this should happen very rarely, since beta-actin is one of most important housekeeping genes for all cell types. In addition, it should be examined whether beta-actin is preferentially degraded (an unlikely event). Another thing to check is whether the secondary antibody is suitable for the particular anti-beta-actin antibody.

This is not advisable. The best approach is to detect the target protein first, then use western blot stripping buffer to remove the target protein antibody and the secondary antibody, and then to incubate with an anti-beta-actin antibody. The reason is that it is difficult to adjust the incubation and to develop parameters for two different antibodies and it is also difficult to prevent cross-reactivity among different antibodies.

The easy way to find the best dilution is to check any information sheet that usually comes with the product. Another approach is to contact the antibody supplier; the supplier usually has optimized the dilution. Regardless, if the suggested dilution does not work, serial dilutions should be tested to find the optimal one.

Multiple experimental procedures may affect this. It could be that the loading solution becomes more sticky, or the transfer is uneven.

Yes. The internal control not only ensures sample loading, but also checks the transfer, incubation, and signal development.

Beta-actin can still be used as an internal control. After the detection of the target protein, the blot should be completely stripped and checked for any signal before incubation with a beta-actin antibody. Alternatively, another protein can be used as an internal control, such as tubulin or GAPDH.

No. The nucleus may contain actin, but its level is dynamic and it should not serve as a control. See Labome's detailed discussion on different loading controls for nuclear sample controls.

There are several reasons: 1) Secondary antibody may not be very specific; 2) The loading buffer is not effective; 3) The beta-actin antibody is not specific; 4) Beta-actin is degraded.