Supporting information
for Moritz et al. “Epicocconone staining: a powerful loading control for Western blots”
Supplementary figures
Supplementary figure 1. Calibration curves of the 6 staining variants with total protein
samples and of E-ToPS with one-protein samples. (A) Same as in Figure 1A, but on a linear X
axis. (B) Background-subtracted and normalized En-ToPS and Es-ToPS signals of ßlactoglobulin plotted against decreasing protein amounts on a linear X axis (3000-0.1 ng; cf.
Fig. 1C; 2-3 replicates each). Error bars depict SEM. (C) Same as B, but with a logarithmic
X axis.
Supplementary figure 2. Biological variation of En-ToPS signals compared to β-tubulin and
GAPDH immunosignals using different brain regions (cochlear nucleus = CN, superior
olivary complex = SOC, inferior colliculus = IC, Rest brain). (A1) En-ToPS of a blot
membrane, depicting 12 samples from the 4 brain regions, each represented by 3 biological
replicates (BR 1-3). Each lane was loaded with 10 µg protein. (A2) Same blot as in A1,
depicting immunosignals for β-tubulin (β-Tub) and GAPDH. (A3) CV%Biol across the 4
regions of β-tubulin, En-ToPS, and GAPDH signals. Lines connect CV% values of the 6
individual BRs.
Supplementary figure 3. Re-evaluation of the calibration curve of En-ToPS from Svensson et
al. [1]. (A) Dilution series of carbonic anhydrase from 1,000-0.06 ng stained with En-ToPS
(from Svensson et al. [1]). White framed rectangles depict areas that we chose for signal
volume measurements. (B) Background-subtracted volumes of the signals in A, plotted
against decreasing protein amounts on a linear X axis. Coefficients of determination for linear
(R2lin) and logarithmic (R2log) regression models are specified. A.u.: arbitrary units. (C) Same
as B, but with a logarithmic X axis. R2log = 0.99 was obtained for a smaller range of 1,000 – 1
ng.
Supplementary Table 1. Costs of the staining compounds
Staining
variant
Commercial name
Distributor
Prize/amount
Used amount/
membrane
Prize/
membrane
En-ToPS
LavaPurple™
Serva Electrophoresis
39€/5mL
31.25 µL
0.24 €
Deep Purple™ Total Protein Stain
GE Healthcare
250€/5mL
31.25 µL
1.56 €
Es-ToPS
SERVA Purple™
Serva Electrophoresis
275€/25mL
31.25 µL
0.34 €
Coomassie
Coomassie R-250
Carl Roth GmbH
69.90€/100g
0.125 g
0.09 €
Sypro Ruby
SYPRO® Ruby protein blot stain
Bio-Rad
200€/200mL
10 mL
10.00 €
β-tubulin
Mouse-anti-β-tubulin T5201
Sigma-Aldrich
314€/200µL
5 µL
7.85 €
GAPDH
Mouse-anti-GAPDH MAB374
Millipore
299€/200µL
10 µL
14.95 €
Supplementary material and methods
Animals, tissue and sample preparation
Organs were obtained from young adult, 8-9-week-old Sprague-Dawley rats. Animal
treatment was in agreement with the German Animal Protection Law and with the NIH guide
for the care and use of laboratory animals. Rats were killed and their organs and brain regions
were prepared as described previously [2] [3].
Tissue was pre-homogenized in a 5-fold weight volume of modified lysis buffer (7 M urea,
2 M thiourea, 2% CHAPS, 30 mM Tris, 100 mM DTT, 4 °C) by 4 strokes in a Teflon/glass
homogenizer and 30 s of sonication. After 10 min lysis at 4 °C, six further strokes were
applied to homogenize the tissue. Lysis was stopped by adding 1/10 volume of 2.5 M sucrose.
Protein concentrations were determined using the Pierce 660 nm Protein Assay (Thermo
Fisher Scientific, Schwerte, Germany).
Electrophoresis (MIAPE-GE-compliant)
Date stamp of initiating step: 2011-05-02. Responsible person: Ralph Reiss, ErwinSchroedinger-Str. 13, 67663 Kaiserslautern, Germany. Electrophoresis type: 1-dimensional.
Samples were incubated under reducing conditions for 5 min at 95 °C. Proteins were
separated in a SDS-PAGE (discontinuous gel; stacking gel: 5% acrylamide, pH 6.8,
82x15x1 mm;
separation
gel:
12%
acrylamide,
pH
8.8,
82x65x1 mm,
acrylamide:bisacrylamide for both gel types: 26.5:1; 15 gel lanes; 3 µL loading buffer: 10%
(v/v) glycerol, 62.4 mM Tris (pH 6.8), 2% (w/v) SDS, 0.01% (w/v) bromophenol blue (final
concentrations); running buffer: 192 mM glycine, 25 mM Tris, 0.1 (w/v) SDS) with 25 mA
(about 1 h; 4 °C) and transferred onto PVDF (Carl Roth GmbH, Karlsruhe, Germany)
membranes for 60 min at 350 mA using tank blotting (Mini Trans-Blot Cell, Bio-Rad,
Munich, Germany).
Total protein staining
Sypro Ruby staining was performed as instructed by the manufacturer. Coomassie staining
was performed as described earlier [4]. E-ToPS (Deep Purple™ Total Protein Stain,
LavaPurple, and SERVA Purple was performed, with marginal modifications, as instructed by
the manufacturers. In brief, the PVDF membranes were incubated for 5 min in deionized
water, followed by another 30 min in 12.5 mL staining solution. After destaining, ethanol
washing, and drying, labeling was visualized. For Sypro Ruby and the E-ToPS variants,
signals were visualized by a VersaDoc 3000 documentation system (Bio-Rad) using 1x1
binning, 4x gain, the 610LP filter and EPI UV illumination.
Immunoblot analysis
The membranes were rehydrated in TTBS and blocked for 30 min in TTBS/milk. In the
latter buffer, it was subsequently incubated with the respective antibodies for 120 min at room
temperature,
anti-glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH)
additionally
overnight at 4 °C.
After four washes in TTBS, the secondary, horseradish peroxidase-conjugated sheep antimouse antibody (NA931, GE Healthcare, Munich, Germany) was applied for 60 min at room
temperature (ca. 22 °C). After four washes in TTBS and one wash in TBS, bound antibodies
were detected utilizing Western Lightning Chemiluminescence Reagent Plus (2x 750 µL;
Perkin Elmer, Waltham, MA, USA) for ß-tubulin and the more sensitive SuperSignal West
Femto Chemiluminescent Substrate (2x 400 µL; Thermo Fisher Scientific) for GAPDH.
Visualisation occurred with the same documentation system as for the total protein stainings
mentioned above; as a modification, 2x2 binning and the chemiluminescence mode was used.
After the immunoreaction for ß-tubulin, blots were not stripped; instead, the
chemiluminescence reaction was stopped in 17% H2O2 for 30 s before incubating with the
primary antibody against GAPDH.
Signal quantification
Quantification of the volume (INT*mm2) of all staining signals (including the reevaluation of Svensson et al. [1]; cf. Suppl. Fig. 3) was performed via Quantity One software
(Version 4.50; Bio-Rad) using equally sized rectangles for the determination of the area of
interest and background subtraction (global method; size- and weight-matched rectangle was
chosen for background).
Calibration curves
Dilution series were made by loading 30-0.0001 µg (Fig. 1) and 2.5-20 µg (Fig. 3) of total
protein of rest brain on different lanes. For normalization, the 30 µg and 2.5 µg signal
volumes, respectively, were set to 100%. The normalized volumes of each were averaged
using the geometrical mean. These averaged normalized volumes of one gel were defined as
one technical replicate. For statistics, 8-11 technical replicates were performed. For assessing
the influence of the E-ToPS on the GAPDH signal (Fig. 3), the 5 µg values were set to 200%,
because the signals obtained at 2.5 µg were too weak.
To check for a significant impact of E-ToPS on subsequent immunostaining, parallel
immunoblots with and without E-ToPS were prepared. The complete procedure (e.g., drying
of the blot membranes, incubation, exposure times) was the same in both approaches. With 5
technical replicates, a paired, two-tailed t-test was used to assess whether the relative impact
([signal volume with E-ToPS divided by signal volume without E-ToPS] multiplied with 100
minus 100) was significantly different from zero.
The regression analysis was performed with the fitting option of Microsoft Excel
(Microsoft Corporation, Redmond, USA; version 14.0.7106.5003).
Image adjustment
For demonstration purpose, contrast of some images (always entire image) was adjusting
with Quantity One. For analysis, the raw figures were used. Appropriate gel and membrane
sections were cropped by using CorelDraw (Version 16.0.0.707). The uncropped and
unadjusted images are shown below, together with the file names and image information.
Uncropped and unadjusted images
Fig. 1A1
2013-07-17 Hg 1S 04_Verdünnungsreihe breit_LavaPurple_4gain_1x1bin_10s_DTT in SB
1643x868 pixel, 72 dpi, 8bit
Fig. 1A1
2013-10-09 Hg1S46_Verdünnungsreihe breit_ServaPurple_4gain_1x1bin_15s_mit DTT
1522x801 pixel, 72 dpi, 8bit
Fig. 1A1
Hg1S17-20 Coomassie037_Represent
1061x868 pixel, 72 dpi, 8bit
Fig. 1A1
2013-08-20 Hg 1S 30_Verdünnungsreihe breit_SyproRuby_4gain_1x1bin_2s_DTT in SB
1643x868 pixel, 72 dpi, 8bit
Fig. 1A2
2013-07-26 Hg 1S 09_Verdünnungsreihe breit_Tubulin 1_1000 _4gain_1x1bin_ 15s_DTT in
SB
1303x868 pixel, 72 dpi, 8bit
Fig. 1A2
2013-07-24 Hg 1S 09_Verdünnungsreihe breit_GAPDH 1_500 SUPER ECL_4gain_1x1bin_
40s_DTT in SB
1551x849 pixel, 72 dpi, 8bit
Fig. 1C
2013-08-27 Lg05_Verdünnungsreihe breit_LavaPurple_4gain_1x1bin_10s_DTT in SB
1643x868 pixel, 72 dpi, 8bit
Fig. 1C
2013-10-25 Lg28_Verdünnungsreihe breit_ServaPurple_4gain_1x1
1643x868 pixel, 72 dpi, 8bit
Fig. 1C
2013-08-27 Lg06_Verdünnungsreihe breit_SYPRO Ruby_4gain_1x1bin_2s_DTT in SB
1643x868 pixel, 72 dpi, 8bit
Fig. 2A1
biorad 2012-07-19 09hr 25min_DP_BRX,XI,XII_18.7._SM.tiff
1303x868 pixel, 72 dpi, 8bit
Fig. 2A2 top
biorad 2012-07-19 14hr 26min-3_ß-Tubulin_BR_X,XI,XII_SM.tiff
1303x868 pixel, 72 dpi, 8bit
Fig. 2A2 bottom
biorad 2012-07-20 10hr 13min_GAPDH_BRX,XI,XII_SM.tiff
1303x868 pixel, 72 dpi, 8bit
Fig. 2B1
2011-08-29_12xRest-Hg-1E5_DeepPurple-2.tiff
1277x868 pixel, 72 dpi, 8bit
Fig. 2B2 top
2011-08-31_12xRest-Hg-1E5_Tubulin.tiff
1293x868 pixel, 72 dpi, 8bit
Fig. 2B2 bottom
2011-08-30_12xRest-Hg-1E5_GAPDH.tiff
1295x868 pixel, 72 dpi, 8bit
Fig. 3A1 top right
2011-09-06_Rest-Hg1E10_Verduennungsreihe_OHNE-DeepPurple_Tubulin.tiff
1297x868 pixel, 72 dpi, 8bit
Fig. 3A1 bottom left
2011-09-06_Rest-Hg1E9_Verduennungsreihe_POST-DeepPurple_Tubulin.tiff
1245x868 pixel, 72 dpi, 8bit
Fig. 3A1 top right
2011-09-06_Rest-Hg1E10_Verduennungsreihe_OHNE-DeepPurple_GAPDH.tiff
1303x868 pixel, 72 dpi, 8bit
Fig. 3A1 bottom right
2011-09-06_Rest-Hg1E9_Verduennungsreihe_POST-DeepPurple_GAPDH.tiff
1303x868 pixel, 72 dpi, 8bit
Suppl. Fig. 1A1
2011-05-02_Hg-Blot1.1_DeepPurple_2x2bin-2.tiff
1620x868 pixel, 72 dpi, 8bit
Suppl. Fig. 1A2 top
2011-07-29_Hg-Blot1.1_beta-Tubulin-wdh.tiff
1303x868 pixel, 72 dpi, 8bit
Suppl. Fig. 1A2 bottom
2011-08-11_Hg-Blot1.1_GAPDH_1-500.tiff
1277x868 pixel, 72 dpi, 8bit
References
[1] Svensson, E., Hedberg, J. J., Malmport, E., Bjellqvist, B., Fluorescent in-gel protein
detection by regulating the pH during staining. Anal. Biochem. 2006, 355, 304-306.
[2] Ehmann, H., Salzig, C., Lang, P., Friauf, E., Nothwang, H. G., Minimal sex differences in
gene expression in the rat superior olivary complex. Hear. Res. 2008, 245, 65-72.
[3] Becker, M., Nothwang, H. G., Friauf, E., Different protein profiles in inferior colliculus
and cerebellum: a comparative proteomic study. Neuroscience 2008, 154, 233-244.
[4] Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., Improved staining of proteins in
polyacrylamide gels including isoelectric focusing gels with clear background at nanogram
sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255262.