SUPPORTING INFORMATION
Tips on improving the efficiency of electrotransfer of target proteins from
Phos-tag SDS-PAGE gel
Emiko Kinoshita-Kikuta, Eiji Kinoshita*, Ayumi Matsuda, and Tohru Koike
Department of Functional Molecular Science, Institute of Biomedical and Health Sciences,
Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan
Corresponding Author *E-mail: kinoeiji@hiroshima-u.ac.jp
Table of Contents
Page
Supporting Information Methods
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Supporting Information Figure 1
S7–S8
Supporting Information Figure 2
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References
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Abbreviations: AP, alkaline phosphatase; BPB, bromophenol blue; EGF, epidermal
growth factor; ERK1/2, extracellular-signal-regulated kinase 1/2; GSK-3, glycogen
synthase kinase 3; HRP–SA, horseradish peroxide-conjugated streptavidin; MEK1,
mitogen-activated protein kinase kinase 1
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Supporting Information Methods
1 Materials
Phos-tag Acrylamide and Phos-tag Biotin (BTL-111), which are commercially available from
Wako Pure Chemical Industries (Osaka, Japan), were synthesized as described previously
[1,2]. ClearTrans SP PVDF membrane and 0.5 M EDTA (pH 8.0) were purchased from Wako
Pure Chemical Industries. Alkaline phosphatase (AP) [from bovine intestinal mucosa,
≥2,000 DEA units/mg protein: one unit will hydrolyze 1.0 µmole of p-nitrophenol phosphate
per minute at 37 °C. Diethanolamine (DEA) units are measured in a 1.0 M diethanolamine
buffer, pH 9.8, containing 0.5 mM MgCl2, substrate concentration 15 mM.], -casein,
-casein, ovalbumin, and pepsin were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Horseradish peroxide-conjugated streptavidin (HRP–SA) was purchased from GE Healthcare
Bioscience (Piscataway, NJ, USA). Anti-glycogen synthase kinase 3 (GSK-3) antibody and
anti-histone H3 antibody were purchased from Cell Signaling Technology (Danvers, MA,
USA). Anti-extracellular-signal-regulated kinases 1/2 (ERK1/2) antibody and anti-Shc
antibody
were
purchased
from
Merck
Millipore
(Darmstadt,
Germany).
Anti-mitogen-activated protein kinase kinase 1 (MEK1) antibody was purchased from BD
Biosciences (San Jose, CA, USA).
2 Preparation of dephosphorylated standard phosphoproteins
For the preparation of completely dephosphorylated -casein, -casein, ovalbumin, and
pepsin, 50 µL of a solution of the corresponding phosphoprotein (10 mg/mL) was mixed with
5.7 µL of an AP reaction buffer [0.5 M Tris–HCl (pH 9) containing 10 mM MgCl2] and 1.3
µL of commercially available AP solution, and the mixture was incubated for 16 h at 37 °C.
To prepare partially dephosphorylated -casein, 20 µL of -casein solution (10 mg/mL) was
mixed with 3 µL of the AP reaction buffer, 6 µL of distilled water, and 1 µL of the AP solution,
and the mixture was incubated for 0, 5, 10, 30, 60, or 120 min at room temperature. In each
case, the reaction was stopped by adding a sample-loading buffer for SDS-PAGE [65 mM
Tris–HCl (pH 6.8), 1% w/v SDS, 10% v/v glycerol, 5% v/v 2-sulfanylethanol, and 0.03% w/v
bromophenol blue (BPB) (all final concentrations)].
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3 Preparation of cell extracts
HeLa cells (107 cells on a 90-mm culture dish) were lysed in 0.5 mL of a lysis buffer [50 mM
Tris–HCl (pH 7.5), 150 mM NaCl, 0.25% w/v deoxycholic acid, 1% v/v NP-40, 1 mM EDTA,
and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)]. The lysate was centrifuged at
14,000g for 10 min, and the supernatant was transferred to a new tube and stored as a sample
of cell extract at –80 °C. The protein concentration was 2 mg/mL. To dephosphorylate the
intracellular phosphoproteins in the cell extract, a 100-µL portion of the extract solution was
mixed with 100 µL of the AP reaction buffer and 10 µL of the commercially available AP
solution, and the mixture was incubated for 2 h.
Cell extracts from A431 and HeLa cells for immunoblotting analyses were prepared as
follows. A431 cells (106 cells on a 35-mm culture dish) were treated in the presence or
absence of 100 ng/mL epidermal growth factor (EGF) for 5 min at 37 °C. HeLa cells (10 6
cells on a 35-mm culture dish) were treated in the presence or absence of 100 nM calyculin A
for 30 min at 37 °C. In addition, HeLa cells (106 cells on a 35-mm culture dish) were
independently treated in the presence or absence of 1 mM pervanadate (a mixture of 1 mM
Na3VO4 and 1 mM H2O2) for 30 min at 37 °C. The treated and untreated cells were washed
with Tris-buffered saline containing 10 mM Tris–HCl (pH 7.5) and 0.10 M NaCl at room
temperature, then lysed with the sample-loading buffer for SDS-PAGE and boiled for 3 min.
4 Phos-tag SDS-PAGE
Phos-tag SDS-PAGE was performed by using a 1-mm-thick, 9-cm-wide, and 9-cm-long gel
on a PAGE apparatus (Atto, model AE-6500, Tokyo, Japan). For Zn2+–Phos-tag SDS-PAGE,
the separating gel (6.3 mL) consisted of 8–10% w/v polyacrylamide and 357 mM
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol and hydrochloric acid
(Bis-Tris–HCl buffer, pH 6.8), and the stacking gel (1.8 mL) consisted of 4% w/v
polyacrylamide and 357 mM Bis-Tris–HCl buffer (pH 6.8). Phos-tag Acrylamide (25–100
µM) and two equivalents of ZnCl2 were added to the separating gel before polymerization.
For Mn2+–Phos-tag SDS-PAGE, the separating gel consisted of 10% w/v polyacrylamide and
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375 mM Tris–HCl buffer (pH 8.8), and the stacking gel consisted of 4% w/v polyacrylamide
and 125 mM Tris–HCl buffer (pH 6.8). Phos-tag Acrylamide (25–100 µM) and two
equivalents of MnCl2 were added to the separating gel before polymerization. An acrylamide
stock solution was prepared containing a 29:1 acrylamide–N,N'-methylenebisacrylamide
mixture. The running buffer for Zn2+–Phos-tag SDS-PAGE consisted of 0.10 M Tris and 0.10
M 3-(N-morpholino)propanesulfonic acid (MOPS) containing 0.10% w/v SDS and 5.0 mM
NaHSO3. The NaHSO3 was dissolved immediately before use. The running buffer for
Mn2+–Phos-tag SDS-PAGE consisted of 192 mM glycine and 25 mM Tris containing 0.10%
w/v SDS. Electrophoresis was performed at 30 mA/gel until the BPB dye reached the bottom
of the separating gel.
5 Semi-dry blotting
Semi-dry blotting was performed by using an NB-1600 blotting system (Nippon Eido, Tokyo,
Japan) operated at 2 mA/cm2 with three transfer buffers (A, B, and C). After Phos-tag
SDS-PAGE, the gel was soaked, with gentle agitation, in transfer buffer B (25 mM Tris and
5% v/v MeOH) with or without 1 mM EDTA for 30 min. The gel was then incubated, with
gentle agitation, in transfer buffer B without EDTA for a further 10–30 min. A PVDF
membrane and six pieces of 3MM paper (Whatman, Maidstone, UK) were trimmed to the
dimensions of the gel. The PVDF membrane was soaked in MeOH for 30 s and then soaked in
transfer buffer B for more than 30 min. Three 3MM papers were soaked in transfer buffer C
(25 mM Tris, 40 mM 6-aminohexanoic acid, and 5% v/v MeOH) and piled on the negative
pole board. The gel, the PVDF membrane, and a 3MM paper soaked in transfer buffer B were
piled up in order. Then, two pieces of 3MM paper soaked in transfer buffer A (0.30 M Tris
and 5% v/v MeOH) were added to the pile. Finally, the pile was covered with the
positive-pole board, and an electric current was supplied for 30 min.
6 Wet-tank blotting
Wet-tank blotting was performed by using a BioRad minitransblot blotting system (Hercules,
CA, USA). After Phos-tag SDS-PAGE, the gel was soaked, with gentle agitation, in a transfer
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buffer (25 mM Tris, 192 mM glycine, and 10% v/v MeOH) with or without 1 mM EDTA for
30 min. The gel was incubated, with gentle agitation, in the transfer buffer without EDTA for
a further 10–30 min. A PVDF membrane and four pieces of 3MM paper were trimmed to the
dimensions of the gel. The PVDF membrane was soaked in MeOH for 30 s and then soaked in
the transfer buffer for more than 30 min. To form a ‘blotting sandwich’ on the electroblotting
screen attached to the blotting system, the gel, the PVDF membrane, and the 3MM papers
were assembled in the transfer buffer as previously described [3]. A voltage (100 V, 4.5 cm
separation of electrode) was applied for 60 min with cooling. For experiments in which a
transfer buffer containing SDS was used, a 10% w/v SDS solution was gently added to the
tank to give a final concentration of 0.1% w/v just before the electricity was switched on.
7 Detection of phosphorylated proteins by Phos-tag Biotin
To prepare the complex of Phos-tag Biotin with HRP–SA, a 10 mM aqueous solution of the
Phos-tag Biotin ligand (10 µL), a 10 mM aqueous solution of Zn(NO3)2 (20 µL), a
commercially available solution of HRP–SA (1 µL), and 469 µL of a solution containing 10
mM Tris–HCl (pH 7.5), 0.10 M NaCl, and 0.10% v/v Tween 20 (TBS-T solution) were mixed
and allowed to stand for 30 min at room temperature as previously described [2]. The mixture
(500 µL) was then placed in the cup of a centrifugal filter device (Spin-X UF 500
Concentrator, 50K MWCO PES, Corning, NY, USA) and centrifuged for 20 min at 14,000g to
remove excess Phos-tag Biotin. The solution (<10 µL) remaining in the reservoir was diluted
with 30 mL of TBS-T solution and used as a probing reagent (Zn2+–Phos-tag-bound HRP–SA
solution). Protein-blotting membranes
soaked in
TBS-T were probed with
the
Zn2+–Phos-tag-bound HRP–SA solution for 60 min, then washed with TBS-T solution. All
ECL images were obtained by using Lumigen TMA-6 (Lumigen, Southfield, MI, USA) and
an LAS 3000 image analyzer (Fujifilm, Tokyo, Japan).
8 Detection of phosphorylated ERK1/2, MEK1, Shc, GSK-3, or histone H3 by
their respective antibodies
After semi-dry blotting, the protein-blotting membrane was soaked in a TBS-T solution for 1
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h, then blocked by treatment with a TBS-T solution containing 1.0% w/v BSA for 1 h. For
immunoprobing analysis of each target protein, the membrane was probed with a solution
containing an appropriate antibody for 1 h. The antibody solutions were prepared by dilution
of the commercially available products with a TBS-T solution. HRP-conjugated anti-mouse
IgG antibody (Cell Signaling Technology) and HRP-conjugated anti-rabbit IgG antibody (Cell
Signaling Technology) were used as secondary antibodies. The membrane probed with each
antibody was washed three times with a TBS-T solution (2.0 ml/cm) for 10 min in each case.
Finally, the target proteins were detected as ECL signals by using Lumigen TMA-6 and an
LAS 3000 image analyzer. The results of antibody-based detection were shown below in
Supporting Information Fig. 1.
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Supporting Information Figure 1
Figure 1. Improvement in the sensitivity of detection of phosphorylated forms derived from
typical intracellular proteins achieved by an additional EDTA treatment in semi-dry blotting
from Zn2+–Phos-tag gel. Extracted proteins from A431 cells (10 µg) treated or not treated
with EGF, and extracted proteins from HeLa cells (10 µg) treated or not treated with calyculin
A or pervanadate were separated by using 25 µM Zn2+–Phos-tag gels. The gels were then
soaked in transfer buffer B with (+) or without (–) 1 mM EDTA, and semi-dry blotting was
performed. Each blotting membrane was probed with the anti-ERK1/2, MEK1, Shc, GSK-3,
or histone H3 antibody. The probing and ECL detection procedures for each pair of blots
(EDTA – and EDTA +) were performed under the same experimental condition at the same
time. Arrows show nonphosphorylated forms of the target proteins.
ERK1/2, MEK1, and Shc are cellular signaling molecules that are phosphorylated within
several minutes of stimulation by EGF. EGF-stimulated and nonstimulated crude extracts
from A431 cells were separated by PAGE on a Zn2+–Phos-tag gel (25 μM) and then subjected
to semi-dry blotting to detect the three signaling molecules. When the Zn2+–Phos-tag gel was
subjected to the EDTA treatment described above, the ECL signals from protein bands in all
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the treated samples (EDTA, +) were stronger than those in the corresponding untreated
samples (EDTA, –). In addition, some bands for phosphorylated forms of the proteins were
not detected in samples not treated with EDTA.
The strengths of signals from phosphorylated forms of GSK-3 and histone H3 increase
when they are treated with the serine/threonine phosphatase inhibitor calyculin A or when
they are treated with the tyrosine phosphatase inhibitor pervanadate. Signals from these
proteins were weak on the blotting membranes obtained from the gel not treated with EDTA,
and some bands corresponding to phosphorylated forms were not detected. Note that in
immunoblotting analyses using antibodies against the respective proteins, both the
EDTA-treated and the untreated membranes were subjected to probing and detection at the
same time and under the same conditions.
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Supporting Information Figure 2
Figure 2. Improvement in the electrotransfer efficiencies of proteins by using a transfer buffer
containing 0.05–0.2% w/v of SDS in wet-tank blotting from Phos-tag gels. Extracted proteins
from HeLa cells (2.0 5.0, and 10 µg) were analyzed by using Laemmli’s SDS-PAGE gels [4]
containing 100 µM Mn2+–Phos-tag (lanes 1, 2, and 3, respectively), followed by wet-tank
blotting with two-ply PVDF membranes. (A) The first membranes (1st blot) faced with
directly Phos-tag gels were stained with CBB R-250 and probed with Zn2+–Phos-tag-bound
HRP–SA solution. All staining, probing, and detection procedures were performed under the
same experimental conditions at the same time. We found that the addition of SDS to the
transfer buffer was effective dose-dependently in enhancing the amounts of total proteins and
phosphoproteins eluting from Phos-tag gels. (B) The second membranes (2nd blot) faced with
the first membranes were stained with CBB R-250 and probed with Zn2+–Phos-tag-bound
HRP–SA solution. All staining, probing, and detection procedures were performed under the
same experimental conditions at the same time. These results demonstrated that the addition
of SDS causes total proteins and phosphoproteins to pass through the first PVDF membrane.
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References
[1] Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., Koike, T., Phosphate-binding
tag, a new tool to visualize phosphorylated proteins. Mol. Cell. Proteomics 2006,
5, 749–757.
[2] Kinoshita, E., Kinoshita-Kikuta, E., Sugiyama, Y., Fukada, Y. et al., Highly
sensitive detection of protein phosphorylation by using improved Phos-tag Biotin.
Proteomics 2012, 12, 932–937.
[3] Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., Separation and detection of large
phosphoproteins using Phos-tag SDS-PAGE. Nat. Protoc. 2009, 4, 1513–1521.
[4] Laemmli, U. K., Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 1970, 227, 680–685.
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