Analytical Biochemistry 401 (2010) 318320

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Analytical Biochemistry
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Notes & Tips

Reversible Ponceau staining as a loading control alternative to actin in Western blots

Isabel Romero-Calvo, Borja Ocn, Patricia Martnez-Moya, Mara Dolores Surez, Antonio Zarzuelo,
Olga Martnez-Augustin, Fermn Snchez de Medina *
Departments of Biochemistry and Molecular Biology II and Pharmacology, School of Pharmacy, University of Granada, Centro de Investigacin Biomdica en Red de Enfermedades
Hepticas y Digestivas (CIBERehd), Campus de Cartuja s/n, 18071 Granada, Spain

a r t i c l e

i n f o

Article history:
Received 11 November 2009
Received in revised form 12 February 2010
Accepted 26 February 2010
Available online 3 March 2010

a b s t r a c t
It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is
the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and
measured actin in parallel under different conditions. Our results show that densitometric analysis is
comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody
probing is validated as an alternative to actin blotting.
2010 Elsevier Inc. All rights reserved.

Gels and blots are one of the fundamental tools of molecular

biology. Their foremost utility is to assess the characteristics of nucleic acids and proteins in electrophoretic movement, indicating
changes in size, glycosylation, dimerization, and so on. However,
they are also frequently used to measure quantitative changes in
gene expression among samples in spite of the known limitations
of this approach, including a small dynamic range and a certain
unpredictability. In particular, the quantitative use of Northern
blots has been classically hampered by the characteristic liability
of RNA because degradation can easily pass for downregulation
in the blot. Hence, parallel measurement of a housekeeping gene,
such as actin, 18S, or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),1 has become commonplace. This procedure is known as
loading control, but this is probably a misnomer given that its primary objective is to rule out degradation, not faulty gel loading, as a
confounding factor; because pipetting is required in virtually every
experiment, this line of reasoning would lead to the use of loading
controls for each one of them.
At any rate, the loading control practice is increasingly extending to Western blots, and it is required by many journals even
though the need for this step is debatable. Ponceau S staining of
proteins has long been applied to quality control of membrane
transfer in Western blotting. Ponceau S staining is fast, inexpensive, and nontoxic; above all, binding is fully reversible in a few
minutes. Here we propose the use of Ponceau S staining as an alternative means to actin immunoblotting to assess equal loading in
Western blots.
To do this, we obtained samples from different organs derived
from two female Wistar rats of approximately 200 g weight. The

* Corresponding author. Fax: +34 958 248964.

E-mail address: fsanchez@ugr.es (F. Snchez de Medina).
1
Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDS,
sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid.
0003-2697/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.02.036

animals were sacriced by cervical dislocation, and colon, liver,

and kidney samples were extracted, homogenized in RIPA buffer
with freshly added protease inhibitors (phenylmethylsulfonyl uoride, aprotinin, leupeptin, pepstatin A, and 1,10-phenanthroline),
cleared by centrifugation, and heated at 95 C for 4 min in denaturing Laemmli buffer. The composition of the RIPA buffer was as
follows: 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, and 1% Triton X-100 in phosphate-buffered saline. The composition of the Laemmli buffer (5) was as follows: 312 nM SDS, 50%
(v/v) glycerol, 1% (v/v) 2-mercaptoethanol, 22.5 mM ethylenediaminetetraacetic acid (EDTA) trisodium salt, 220 mM Tris, and
traces of bromophenol blue (pH 6.8). Protein content was measured by the bicinchoninic acid method using bovine serum albumin as standard [1]. Then we ran a series of Western blots with
varying loading conditions and assessed gel loading either by Ponceau S staining (right after transfer) or by actin immunoblotting
using the JLA-20 monoclonal antibody. The JLA-20 antibody against
actin developed by Lin [2] was obtained from the Development
Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and
maintained by the Department of Biological Sciences at the University of Iowa. The secondary peroxidase-conjugated antibody was
obtained from Sigma (Barcelona, Spain). We used Bio-Rad electrophoresis and transfer units and ScheringPlough nitrocellulose
membranes with 90-min transfer time and 100-V constant voltage
settings. After transfer, the nitrocellulose membranes were rinsed
briey in distilled water and incubated in Ponceau S solution (0.5
[w/v] in 1% [v/v] acetic acid) for 2 min [3], followed by a brief rinse
in distilled water so that the lanes and bands were clearly visible.
The membranes were then inserted in between transparency
sheets and scanned at 300 dpi to a TIFF le using a standard scanner (Epson Perfection 3490 Photo). After that, the membranes were
rinsed once more in distilled water for 23 min until the staining
was completely eliminated, and we proceeded with the blocking

Notes & Tips / Anal. Biochem. 401 (2010) 318320

and antibody incubation steps as usual. Antibody-bound peroxidase was detected by enhanced chemiluminescence (PerkinElmer
Life Sciences, Boston, MA, USA), documented in Kodak lm, and
quantitated with Scion Image software. Because Ponceau S is a
nonspecic protein dye, all proteins in the membrane are colored.
We selected a section of the blot that covered a wide range of
molecular weights in each case, typically approximately 90% or
more of the lane length.
We initially tested the capacity of reversible Ponceau S staining
to assess equal gel loading and, accordingly, ran a series of gels featuring the different tissue samples at equal levels of protein loading, namely at 140, 80, 40, and 10 lg of protein per lane, thereby
covering the entire range of small gels. Fig. 1 shows a typical
Ponceau stain and actin signal. Clearly, the results are very similar
with both methods. As expected, there are no differences depending on the organ considered (not shown). However, the slopes do
differ depending on the organ, so that correlation for each individual type of sample is typically around 0.9 (r2), whereas linearity is
impaired in the graph showing the combined data.
As alluded to earlier, Western blots may be used for quantitative purposes, although this approach has well-acknowledged limitations, particularly in terms of the dynamic range, that is, the
difference between the threshold signal that is detectable and
the saturation point beyond which the signal is not further increased or is increased only marginally even though the protein
detected may be present in much higher amounts. Thus, we set
out to verify whether the dynamic range is different for Ponceau
S and actin immunoblotting. The analysis of the densitometric
quantitation of the Ponceau S and actin signals is shown in Fig. 2.
The results are reasonably linear up to 140 lg of protein loaded
in both cases, indicating that there is no limitation of dynamic

Fig. 2. Dynamic range of actin and Ponceau S densitometric

(circles) and liver (squares) samples in different quantities
analyzed by Western blot, including reversible Ponceau S
incubation with primary antibody. Both actin (solid lines) and
lines) densitometric signals are shown.

319

signal. Rat kidney

(10140 lg) were
staining prior to
Ponceau S (broken

range imposed by the use of Ponceau S compared with actin. Actually, the linearity was enhanced with Ponceau S in this particular
experiment, as indicated by the higher difference between the
maximal and minimal densitometric values. It should be noted,
however, that Western blotting has well-known limitations for
quantitative purposes either way.
Our data provide validation for the use of reversible Ponceau S
staining to assess equal gel loading, or quality control, in Western
blots. Certainly, there are limitations to our validation. In principle,
it applies only to the conditions used (i.e., rat colon, kidney, and liver). We have also studied mouse organs, as well as HT29, T84, and
Caco-2 human cells, with similar results. Moreover, our method
can be extended to any other type of sample with minimal effort.
For instance, it seems to work equally well for bone and jejunum
rat samples (although this was not tested extensively). The same
applies to technical conditions in our study such as the use of
nitrocellulose membranes, blocking buffer, and antibodies.
Ponceau staining has an additional advantage in that it does not
rely on a single protein for normalization or loading control. This
circumvents the possibility that the housekeeping proteins used
for this purpose may actually vary in some conditions or that they
are saturated at the levels of loading necessary for detection of
low-expression products [4,5].
In conclusion, we have shown that reversible Ponceau S staining
can be used advantageously over actin detection for quality or
equal loading control in Western blotting. It is equally useful for
this purpose, has similar or improved dynamic range, is extremely
inexpensive, and takes no longer than 10 min.
Acknowledgments
This work was supported by the Ministerio de Ciencia e Innovacin (SAF2008-01432 and AGL2008-04332). I.R.-C. and P.M.-M. are
funded by the Ministerio de Ciencia e Innovacin. The Centro de
Investigacin Biomdica en Red de Enfermedades Hepticas y
Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III.
References

Fig. 1. Correlation between Ponceau S and actin densitometric signal. Rat colon,
kidney, and liver samples were analyzed by Western blot, including reversible
Ponceau S staining prior to incubation with primary antibody.

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