2844 Electrophoresis 2006, 27, 2844–2845

Angela Dittmer Short Communication

Jürgen Dittmer

Klinik für Gynäkologie, â-Actin is not a reliable loading control in

Universität Halle,
Halle, Germany
Received October 18, 2005
Accepted December 17, 2005
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, anti-
bodies 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

Western blot analysis is a commonly used method to
semiquantitatively measure the expression and phos-
phorylation status of proteins by specific antibodies [1–4].
Proteins are denatured, separated in an SDS-polyacryl-
amide 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 abun-
dant 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 Signal-
ing #4967), caveolin-1 (Cav-1, 1:2500, 1:5000, or
Correspondence: Dr. Jürgen Dittmer, Klinik für Gynäkologie, Univer-
sität 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-3-
phosphate 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 con-
jugate (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 con-
trast, 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 anti-
GAPDH antibody overnight. Further dilution of the poly-
clonal 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 cor-
responding Western blot membranes with the two anti-b-
actin antibodies and the anti-GAPDH antibody for 1 h.

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Electrophoresis 2006, 27, 2844–2845
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.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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 anti-
body 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-b-
actin antibody, no change in band intensity could be
observed in the range of 7.5–1.88 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-b-
actin 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 incu-
bation 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 differ-
ences in the level of its target protein.
References
[1] Dennis-Sykes, C. A., Miller, W. J., McAleer, W. J., J. Biol.
Stand. 1985, 13, 309–314.
[2] Rybicki, E. P., von Wechmar, M. B., J. Virol. Methods 1982, 5,
267–278.
[3] Towbin, H., Staehelin, T., Gordon, J., Proc. Natl. Acad. Sci.
USA 1979, 76, 4350–4354.
[4] Vetter, M., Blumenthal, S. G., Lindemann, R. K., Manns, J. et
al., Oncogene 2005, 24, 650–661.
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