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Analytical Biochemistry
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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.
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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
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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.
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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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