Analytical Biochemistry 467 (2014) 1–3

Contents lists available at ScienceDirect

Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Notes & Tips

Stain-free detection as loading control alternative to Ponceau and

housekeeping protein immunodetection in Western blotting
B. Rivero-Gutiérrez a,1, A. Anzola a,1, O. Martínez-Augustin b, F. Sánchez de Medina a,⇑
a
Department of Pharmacology, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación
Biosanitaria (ibs.GRANADA), University of Granada, 18071 Granada, Spain
b
Department of Biochemistry and Molecular Biology II, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de
Investigación Biosanitaria (ibs.GRANADA), University of Granada, 18071 Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: It is currently a routine practice to require a measurement of a housekeeping reference, including actin,
Received 11 June 2014 glyceraldehyde-3-phosphate dehydrogenase, b-tubulin, among others, in Western blots, as it is the rule in
Received in revised form 13 August 2014 RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here
Accepted 22 August 2014
we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and house-
Available online 1 September 2014
keeping protein immunodetection under different conditions. Our results show that Stain-Free gels out-
perform Ponceau staining and that both are more consistent than housekeeping proteins as a loading
Keywords:
control.
Western blot
Loading control
Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
Ponceau staining creativecommons.org/licenses/by/3.0/).
Housekeeping proteins
TGX Stain-Free gels

The use of so called ‘loading controls’ is standard practice in RNA the absence of changes in housekeeping protein expression is nor-
gels [1]. Despite the name, it is not equal loading but the absence of mally assumed rather than established. In some cases these proteins
RNA degradation that is sought by this approach. This is based on are indeed affected by the experimental conditions, for instance GAP-
the well-known liability of RNA, but it has gradually extended also DH, b-actin and b2-microglobulin in normal vs. inflamed intestine,
to the technical validation of Western blots, despite the fact that GAPDH in Von Hippel Lindau defective or transfected cell lines, and
proteins are much more stable, especially after processed for elec- again GAPDH in melanoma vs. control patients, to mention a few doc-
trophoresis. Even if this requirement may be not justified, it has umented examples [2–5]. Because of the relatively high expression
become a common practice. Following again the Northern blot path, level of these proteins and the limited dynamic range of antibodies
most laboratories measure a reference protein to ensure equal load- used in immunodetection, the signal is prone to saturate the detection
ing of different lanes in the protein gel, in effect performing a second system, so that effective changes in expression may not be observed.
parallel Western, preferably in the same blot but sometimes using a Tracking the total protein level after gel loading may be a more
duplicate [2]. To this end, proteins with a substantial expression suitable approach to assess technical correctness [5]. We previ-
level that is considered to be unaffected by the experimental condi- ously applied reversible Ponceau staining of protein blots to track
tions in the study (i.e. ‘housekeeping’ proteins) are typically used, the total protein level in the blot lanes and compared this tech-
usually actin, glyceraldehyde-3-phosphate dehydrogenase (GAP- nique with housekeeping protein normalization [6]. Ponceau stain-
DH)2 or b-tubulin, but also including a variety of other proteins such ing was found to be a low cost, valid alternative in this setting.
as COX-IV (mitochondrial loading control protein), phosphoribosyl- Here we compared both approaches to a new technology intro-
transferase, or cyclophilin, among others. Apart from the obvious duced by Bio-Rad, through TGX Stain-Free gels [7]. The principle
waste of time and resources, a major caveat of this approach is that is based on the fluorescent detection of tryptophan residues con-
tained in the protein sequence which are previously modified by
⇑ Corresponding author at: Department of Pharmacology, CIBERehd, School of a trihalo compound included in the electrophoresis gel after sepa-
Pharmacy, Instituto de Investigación Biosanitaria (ibs.GRANADA), University of ration. Therefore it requires no extra addition of reagents, provided
Granada, Campus de Cartuja s/n, 18071 Granada, Spain. Fax: +34 958 248964. that the trihalo compound containing gels is used.
E-mail address: fsanchez@ugr.es (F.S. de Medina). Female Wistar rats and Zucker obese and lean control rats were
1
Both of these authors contributed equally to this work.
2 used in this study, in addition to Caco-2 cells cultured in a standard
Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDS,
sodium dodecyl sulfate; PVDF, polyvinylidene fluoride; ECL, enhanced chemilumi- fashion. Liver samples and Caco-2 cells were homogenized in RIPA
nescence; CV, coefficient of variation. buffer and processed for Western blot [6]. We ran a series of protein

http://dx.doi.org/10.1016/j.ab.2014.08.027
0003-2697/Ó 2014 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
2 Notes & Tips / Anal. Biochem. 467 (2014) 1–3

Fig.1. Assessment of protein quantity using increasing amounts of rat liver lysate. (A) Total protein detected by Stain-Free (SF) technology in the gel (left) and after transfer
(center), and detected by Ponceau Red staining (right). (B) Immunoblot of the housekeeping proteins GAPDH and actin (images shown in A correspond to the GAPDH blot). (C,
D) Protein quantitation.

blots using TGX Stain-Free gradient precast gels (Bio-Rad). Gel load- reproducibility of about 8% (8.5 ± 0.4%, n = 7) in the gel post elec-
ing control was done by Ponceau S staining (right after transfer), by trophoresis. This was fairly preserved after transfer (9.7 ± 2.3%,
Stain Free detection (in the gel after the electrophoresis and on the n = 7), but was occasionally higher (up to 20%) depending on
blotting membrane after transfer) and by actin or GAPDH immuno- the homogeneity of the transfer across the blot membrane. Red
detection using JLA-20 and GA1R monoclonal antibodies, respec- Ponceau staining includes a manual step by rinsing the excess of
tively. A ChemiDoc™ MP imager (Bio-Rad) was used for gel/blot stain before taking an image. This step is critical as it may generate
documentation in all cases. Antibodies were supplied by the Devel- heterogeneity of staining across the surface of the membrane. In
opment Studies Hybridoma Bank at the University of Iowa (actin), our hands this produced a variability of 13.0 ± 2.9% (n = 7), which
Epitope Biotech Inc. (GAPDH), Cell Signaling Technology (phospho- was the result of increased heterogeneity among blots rather than
specific AKT), BD Biosciences (PCNA) and Sigma (secondary peroxi- of higher signal spread in each blot. Immunodetection of actin and
dase conjugated antibody). We used Bio-Rad electrophoresis and GAPDH in parallel blots showed a %CV of 15.3% and 20.3%, respec-
transfer units and Bio-Rad PVDF Turbo transfer packs, with 10 min tively. Thus a variability of 15–20% is expected with either of the 3
transfer time (25 V and 2.5 A, Trans-Blot Turbo system). After trans- techniques but tends to be lower with Stain Free.
fer, a Stain-Free image was taken and the PVDF membranes were The dynamic range and linearity of the different techniques
rinsed briefly in distilled water and incubated in Ponceau S solution were evaluated by measuring the signal of duplicate lanes of the
(0.5% w/v in 1% v/v acetic acid) for 2 min [3], followed by a brief rinse same sample (rat liver) loaded with increasing amounts of protein.
in distilled water, so that the lanes and bands were clearly visible. The Stain-Free and Red Ponceau techniques showed similar linear
After scanning, the membranes were rinsed once more in distilled range of detection from 5 to 80–120 lg of total proteins that is
water for 2–3 min, until the staining was completely eliminated, compatible with the charge required to detect common low abun-
and we proceeded with the blocking and antibody incubation steps. dant target protein, although linearity was more consistent with
Antibody bound peroxidase was detected by enhanced chemilumi- Stain-Free since some of the blots stained with Red Ponceau
nescence (Clarity™ Western ECL Substrate, Bio-Rad), documented showed saturation (Fig. 1). Conversely, the signal corresponding
and quantitated (ImageLab™ software, Bio-Rad). to GAPDH immunodetection was saturated at 40 lg of protein
In order to assess internal reproducibility, the variation of signal loaded, due to the high sensitivity of this particular antibody. Actin
generated by a single sample (rat liver) loaded in equal amounts in immunodetection was fully linear though, owing to a less strong
different lanes of a gel was measured as the coefficient of variation signal. Similar results were obtained with Caco-2 cells (human
(%CV), defined as the percent standard deviation:mean ratio. It is intestinal epithelial phenotype, Fig. 2).
important to note that Ponceau staining is carried out on the pro- Finally, the three techniques were applied to the study of differ-
tein blot as a direct, one step procedure, while immunostaining ent samples, namely rat liver from obese Zucker rats and their lean
involves a number of washing and antibody incubation steps, plus controls, run in duplicate in the same gel. In this case the intensity
the detection itself, in this case by ECL. In contrast, the Stain-Free of the signal of the different samples was very similar using either
signal can be detected not only in the blot but also in the gel itself. Stain-Free or Red Ponceau detection, consistent with equal loading.
Predictably, the variability increases as the number of steps before However, GAPDH levels were markedly influenced by the type of
detection accumulates. Thus the Stain-Free showed an internal sample, with higher levels being detected in the liver samples
 Notes & Tips / Anal. Biochem. 467 (2014) 1–3 3

Fig.2. Putative housekeeping protein expression may be modified by experimental conditions. Duplicate samples of increasing amounts of Caco-2 lysate were run and
analyzed by Stain-Free, Ponceau staining and PCNA, Actin or GAPDH immunoblotting. (A) Total protein detected by Stain-Free technology in the gel (left) and after transfer
(center), and detected by Ponceau Red staining (right), n = 6. (B) Immunoblot of the putative housekeeping proteins Actin (center) and GAPDH (right), and of PCNA (left), n = 2
for each one (images shown in A correspond to the GAPDH blot). (C) Protein quantitation.

obtained from obese rats compared with their lean controls (not 22812), and by funds from Junta de Andalucía (CTS6736 and
shown). Thus when the signal of a target immunodetected protein CTS164). B. Rivero and A. Anzola were supported by fellowships of
such as phospho-AKT was assessed in a parallel blot, appropriate the Ministry of Education and Science of Spain and Junta de And-
normalization was obtained with either Red Ponceau or Stain-Free, alucía, respectively. CIBERehd is funded by the Instituto de Salud
but not with GAPDH. In short, GAPDH is not a valid housekeeping Carlos III.
protein in this setting and its use can be misleading.
Based on our results, both Stain Free and Red Ponceau total pro- References
tein signals outperform housekeeping protein immunodetection as
[1] F. Herrera, V. Martin, I. Antolin, G. Garcia-Santos, J. Rodriguez-Blanco, C. Rodriguez,
loading controls since (1) there is no added advantage of using Standard curve for housekeeping and target genes: specific criteria for selection of
these compared to Stain-Free or Ponceau (which save extra money loading control in Northern blot analysis, J. Biotechnol. 117 (2005) 337–341.
and time and gives reliable results); (2) antibodies giving a high [2] R.E. Ferguson, H.P. Carroll, A. Harris, E.R. Maher, P.J. Selby, R.E. Banks,
Housekeeping proteins: a preliminary study illustrating some limitations as
signal predictably saturate at higher protein loads; and (3) individ- useful references in protein expression studies, Proteomics 5 (2005) 566–571.
ual loading control proteins are subject to variation in certain bio- [3] G. Bamias, D. Goukos, E. Laoudi, I.G. Balla, S.I. Siakavellas, G.L. Daikos, S.D. Ladas,
logical situations. Stain Free appears to have an increased linear Comparative study of candidate housekeeping genes for quantification of target
gene messenger RNA expression by real-time PCR in patients with
range and less variability than Ponceau staining, the latter proba- inflammatory bowel disease, Inflamm. Bowel Dis. 19 (2013) 2840–2847.
bly derived from the lack of the manual washing step. Conversely, [4] O. Giricz, J.L. Lauer-Fields, G.B. Fields, The normalization of gene expression data
Ponceau staining is cheaper although it is likely that Stain-Free in melanoma: investigating the use of glyceraldehyde 3-phosphate
dehydrogenase and 18S ribosomal RNA as internal reference genes for
technology will also become increasibly affordable over time. quantitative real-time PCR, Anal. Biochem. 380 (2008) 137–139.
Increased range may be of importance when using high protein [5] S.L. Eaton, S.L. Roche, M. Llavero Hurtado, K.J. Oldknow, C. Farquharson, T.H.
loads, in the vicinity of 70–80 lg or higher. These data are in agree- Gillingwater, T.M. Wishart, Total protein analysis as a reliable loading control
for quantitative fluorescent Western blotting, PLoS ONE 8 (2013) e72457.
ment with the report by Colella et al. [8], which showed that Stain
[6] I. Romero-Calvo, B. Ocon, P. Martinez-Moya, M.D. Suarez, A. Zarzuelo, O.
Free is superior to Sypro Ruby protein staining, and also with the Martinez-Augustin, F.S. de Medina, Reversible Ponceau staining as a loading
more recent paper by Gilda and Gomes [9]. control alternative to actin in Western blots, Anal. Biochem. 401 (2010) 318–320.
[7] A. Posch, J. Kohn, K. Oh, M. Hammond, N. Liu, V3 stain-free workflow for a
practical, convenient, and reliable total protein loading control in Western
Acknowledgments Blotting, J. Vis. Exp. 30 (2013), http://dx.doi.org/10.3791/50948.
[8] A.D. Colella, N. Chegenii, M.N. Tea, I.L. Gibbins, K.A. Williams, T.K. Chataway,
Comparison of stain-free gels with traditional immunoblot loading control
The authors want to thank the input of Arnaud Remy and Enrique
methodology, Anal. Biochem. 430 (2012) 108–110.
Orozco of Bio-Rad Laboratories. This study was supported by the [9] J.E. Gilda, A.V. Gomes, Stain-free total protein staining is a superior loading
Ministry of Science and Innovation (SAF2011-22922 and SAF2011- control to beta-actin for Western blots, Anal. Biochem. 440 (2013) 186–188.