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2844 Angela Dittmer Jrgen Dittmer

Klinik fr Gynkologie, Universitt Halle, Halle, Germany

Electrophoresis 2006, 27, 28442845

Short Communication

-Actin is not a reliable loading control in Western blot analysis


b-Actin is often used as a loading control in Western blot analysis. We analyzed the ability of b-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, b-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that b-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein. Keywords: b-Actin / GAPDH / Western blot DOI 10.1002/elps.200500785

Received October 18, 2005 Accepted December 17, 2005

Western blot analysis is a commonly used method to semiquantitatively measure the expression and phosphorylation status of proteins by specific antibodies [14]. Proteins are denatured, separated in an SDS-polyacrylamide gel, and transferred onto a protein-absorbing membrane which then is incubated with the antibody against the protein desired. For each antibody, titration experiments are necessary to optimize S/N. Similarly important is the control for equal protein loading. This is usually done by reprobing the Western blot membrane with an antibody that recognize a protein with relatively constant expression. For this purpose, an anti-b-actin antibody is often used. However, b-actin is highly abundant in cells in contrast to most of the other proteins that are analyzed by Western blot analysis. For this reason, we explored whether the b-actin control is sensitive enough to detect differences in protein loading. To address this issue, several consecutive dilutions of a cytosolic extract of MDA-MB-231 cells, containing 15, 7.5, or 3.75 mg protein, respectively, were separated on 10% SDS-polyacrylamide gel by using a Protean Minigel apparatus (BioRad) and the proteins transferred to a polyvinyldifluoride membrane (Millipore). After blocking the membranes with nonfat milk, the membranes were incubated with polyclonal rabbit antibodies directed against b-actin (1:000, 1:2000, or 1:4000, Cell Signaling #4967), caveolin-1 (Cav-1, 1:2500, 1:5000, or
Correspondence: Dr. Jrgen Dittmer, Klinik fr Gynkologie, Universitt Halle, Ernst-Grube-Str. 40, D-06120 Halle, Germany E-mail: juergen.dittmer@medizin.uni-halle.de Fax: 149-345-557-5261 Abbreviations: ERK, extra cellular-signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Cav-1, caveolin-1

1:10000, BD Transduction Laboraties, #610059), ERK1/2 (ERK = extracellular-signal-regulated kinase; 1:1000, Cell Signalling, #9102), Ets1 (1:2000, Santa Cruz, C-20), with monoclonal mouse antibodies against glyceraldehyde-3phosphate dehydrogenase (GAPDH; 1:5000, Ambion, #4300) or b-actin (1:10000, Sigma, Clone AC-74). The incubation time was either 15 min, 1 h at RT or overnight at 47C. For the chemiluminescent visualization of the antigen/antibody interaction, membranes were treated with anti-rabbit or anti-mouse antibody peroxidase conjugate (GE-Amersham), incubated with ECL plus reagent (GE-Amersham) and light emission detected by exposing the membrane to a Hyperfilm ECL (GE-Amersham). When the membrane was incubated with the antibody against Cav-1, ERK1/2, Ets1, or GAPDH for 15 min, a clear gradual decrease in band intensity along with the decreasing protein load was observed (Fig. 1). In contrast, no or little protein load-dependent reduction in band intensity was found when the polyclonal or monoclonal b-actin antibody was used. Longer incubation times with the primary antibody generally diminished the differences in signal intensities between lanes with different protein loads. Essentially no differences could be observed when membranes were treated with the anti-b-actin antibodies for 1 h or overnight and also with the anti-Cav-1 or antiGAPDH antibody overnight. Further dilution of the polyclonal anti-b-actin to 1:2000 or 1:4000 or anti-Cav-1 to 1:5000 or 1:10000 had little effect on the ability of these antibodies to distinguish between different protein loads. In a second set of experiments, we further serially diluted the protein load down to 0.06 mg and incubated the corresponding Western blot membranes with the two anti-bactin antibodies and the anti-GAPDH antibody for 1 h.
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2006, 27, 28442845

General

2845

Figure 2. Same extract as used for the experiments described in Fig. 1 was further diluted down to 0.06 mg protein and Western blot analyses were carried out with b-actin and GAPDH-specific antibodies.

Signal intensity as generated by the anti-GAPDH antibody again gradually decreased from 7.5 to 0.94 mg of total protein loaded, while no signal could be observed below 0.94 mg (Fig. 2). In the case of the polyclonal anti-bactin antibody, no change in band intensity could be observed in the range of 7.51.88 mg of total protein. At 0.94 mg protein the signal gradually decreased and was undectable at 0.12 mg or lower. The monoclonal anti-bactin antibody generated a similar picture, except that similar band intensities were found in a broader range of protein loads between 0.47 and 7.5 mg. These data indicate that b-actin is not a suitable control protein to check for equal protein loading in Western blot analysis. Instead, GAPDH could be used for this purpose. The data also demonstrate that generally, at longer incubation times, primary antibodies may fail to differentiate between different levels of their target protein. Based on our data, we recommend that, for each individual Western blot analysis, different dilutions of a positive control extract be run along with the samples in the same gel. In this way, it can be judged whether the particular antibody under the conditions as used was able to pick up differences in the level of its target protein.

References
Figure 1. Different amounts of an MDA-MB-231 breast cancer protein extract were analyzed by Western blot analysis by using antibodies directed against b-actin, caveolin-1 (cav-1), ERK1/2, Ets1, or GAPDH. Incubation with these primary antibodies varied between 15 min, 1 h, or overnight.
[1] Dennis-Sykes, C. A., Miller, W. J., McAleer, W. J., J. Biol. Stand. 1985, 13, 309314. [2] Rybicki, E. P., von Wechmar, M. B., J. Virol. Methods 1982, 5, 267278. [3] Towbin, H., Staehelin, T., Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 43504354. [4] Vetter, M., Blumenthal, S. G., Lindemann, R. K., Manns, J. et al., Oncogene 2005, 24, 650661.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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