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A sensitive and reversible staining of proteins on blot membranes
Journal Pre-proof A sensitive and reversible staining of proteins on blot membranes Jun-Ling Wang, Zhao Li, Mei-Qi Li, Wei-Guang Chen, Chao-Jin Xu PII:
S0003-2697(19)31082-6
DOI:
https://doi.org/10.1016/j.ab.2020.113579
Reference:
YABIO 113579
To appear in:
Analytical Biochemistry
Received Date: 1 November 2019 Revised Date:
4 January 2020
Accepted Date: 6 January 2020
Please cite this article as: J.-L. Wang, Z. Li, M.-Q. Li, W.-G. Chen, C.-J. Xu, A sensitive and reversible staining of proteins on blot membranes, Analytical Biochemistry (2020), doi: https://doi.org/10.1016/ j.ab.2020.113579. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
1
A sensitive and reversible staining of proteins on blot membranes
2
Jun-Ling Wang1, Li-Zhao2, Mei-Qi Li2, Wei-Guang Chen3#, Chao-Jin Xu3#
3
1
4
University, Wenzhou, Zhejiang 325000; School of 2nd clinical medical sciences,
5
Wenzhou Medical University, Wenzhou, Zhejiang 325035; 3Department of Histology
6
& Embryology, School of Basic Medical Science, Wenzhou Medical University,
7
Wenzhou, Zhejiang 325035, P.R. China
8
#
9
Correspondence to: Dr Chao-Jin Xu, Department of Histology & Embryology, School
10
of Basic Medical Science, Wenzhou Medical University, Cha Shan university town,
11
No.1 Central North Road, Zhejiang 325035, P.R. China
12
Chao-Jin Xu E-mail: [email protected]
13
Wei-Guang Chen E-mail: [email protected]
14
Wang Jun-Ling Email: [email protected]
15
Li-Zhao Email: [email protected]
16
Mei-Qi Li Email: [email protected]
17
Number of Pages: 11
18
Number of Figures: 7
19
Number of Tables: 1
Center for Reproductive Medicine, Affiliated Hospital 1 of Wenzhou Medical
Co- Correspondence author
20 21 22 23 24 25 26 27 28 29 1
1
Abstract
2
A modified, sensitive and reversible method for protein staining on nitrocellulose
3
(NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western
4
blotting. The method employed Congo red staining to visualize proteins on different
5
blot membranes. Staining of proteins with Congo red dye is more faster procedures.
6
According to the experimental results, approximate 20ng proteins could be detected in
7
3 min in room temperature. The staining on the proteins is easily reversible with
8
Congo red destaining solution for NC and PVDF membranes, so that the blot
9
membranes can be reused for Western blotting. In addition, we confirmed that the
10
staining method is fully compatible with Western blot detection. NC and PVDF
11
membranes treatment with Congo red staining does not interfere with conventional
12
chemiluminescent substrates of peroxidase. As compared to MemCode reversible
13
protein stain kits from Pirece Biotechnology, the staining technique is more sensitive,
14
lower of cost, convenient and not adversely affecting subsequent Western blotting
15
results. On the other hand, the stain is more sensitive than the Ponceau S staining.
16
Therefore, Congo red staining is a promising and ideal alternative for current protein
17
stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated
18
using various 2D and 3D molecular docking and demonstrated potential molecular
19
basis for sensitivity of Congo red staining are higher than Ponceau S.
20 21
Key words: Protein stain; Congo red; Nitrocellulose and PVDF membranes; Western
22
blotting
23 24
Introduction
25
The western blot (sometimes called the protein immunoblot) is a widely used
26
analytical method for visualizing proteins transferred from gels to nitrocellulose,
27
PVDF (polyvinylidenedifluorid) membranes or other immobilized matrices. Although
28
abundant data are available about protein staining by using of different staining agents
29
on the level of polyacrylamide gels, much less is known on the level of blotting
30
membranes [8]. Towbin et al. firstly described the technique of detecting proteins on 2
1
blot membranes [10]. To stain proteins bound to blot membranes, a series of organic
2
dyes have been developed, such as amido black, Ponceau S, Coomassie blue, colloidal
3
silver/gold, India ink, ferridye, Congo red , MemCode and fluorescent dye, SYPRO
4
Ruby [1, 4]. To my knowledge, of proteins staining, Ponceau S, MemCode, and
5
SYPRO Ruby are directly compatible with subsequent immunoblotting [1, 4]. But
6
some stains, such as Coomassie blue, India ink and amido black, are the least
7
sensitive and not well support compatibility [3, 4, 14]. In addition, it is necessary for
8
an ideal protein stain would be high-specific affinity for proteins and no affinity for
9
other biological material. The dye reagents also should bound to proteins strongly and
10
rapidly under normal application conditions, as well as are commercially available.
11
Further, the stains should be compatible with a variety of blot membranes [1]. It is,
12
therefore, our aims was to develop a highly sensitive and less expensive protein
13
staining method toward all blot membranes. During the process of exploration and
14
based on previously research [7], we unexpectedly discovered that Congo red (Fig.1)
15
staining proteins on NC and PVDF membranes quickly with a sensitivity of detection
16
(about 20ng) comparable to that of Ponceau S. The method is simple and can
17
visualize the protein bands on the blot membranes within 3min in the staining
18
working solution. Staining of transferred proteins (～20ng/band) appear as dark
19
blue-violet bands on a white background after rinsing with DW. Besides, removing
20
the dyes from the blot takes about two times at 10minute intervals in destaining
21
solution for subsequent immunological detection. Additionally, sensitivity of detecting
22
proteins on blotting membranes can be up to around 25-fold than previous Congo red
23
staining protocol.
24
All in all, the stain is more sensitive than the Ponceau S stain and much cheaper
25
than MemCode with the properties of an ideal stain. Consequently, we conclude that
26
Congo red staining should be the preferred reversible staining after proteins transfer in
27
future Western blot approaches.
28
Materials and methods
29
Materials
30
PageRuler™ Unstained Protein Ladder was from Thermo Fisher Scientific 3
1
Biotechnology. Dual Color Prestained Protein Marker was from Epizyme
2
Biotechnology. Congo red ( Sigma-Aldrich); His-Tag Mouse Monoclonal Antibody
3
(Affinity
4
Biotechnology); p44/42 MAPK (Erk1/2) rabbit mAb (Cell Signaling Technology);
5
HRP-conjugated goat anti-rabbit IgG (Beyotime Biotechnology). Ponceau 3R were
6
from Sigma-Aldrich. Nitrocellulose membrane (NC) and Polyvinylidenefluoride
7
(PVDF) and BeyoECL Star chemiluminescent substrate were from Beyotime
8
Biotechnology. Methanol (Changshu Hongsheng Fine Chemical Co., Ltd; China).
9
Neural stem cells (NSCs) were prepared from embryonic day 17 (E17) SD rats
10
(supplied by Chinese Academy of Sciences) according to the protocols established by
11
our group [12].
12
Western blot analysis
Biosciences);
HRP-conjugated
goat
anti-mouse
IgG
(Beyotime
13
Thermo Scientific Unstained Protein Molecular Weight Marker containing
14
several native proteins (size from 10 to 200 kDa), NSCs lysate proteins, and purified
15
His-NogoA△20 protein (amino acid peptide 564-749,~23KD; unpublished data)
16
were used as model proteins. Proteins were separated on 5~10% Tris–glycine SDS–
17
PAGE gels and transferred onto nitrocellulose or PVDF membranes, according to the
18
methods established by us [12, 13]. The total protein concentration was measured by
19
Detergent Compatible Bradford Protein Assay Kit (Beyotime Biotechnology). Images
20
were acquired on the Clinx imager (www.clinx.cn).
21
Staining, destaining, and stain erasing procedure for NC and PVDF membranes
22
After electro blotting, the NC and PVDF membrane were rinsed with deionized
23
water (DW) for 2 x 5min, and stained with Congo red staining solution for 3 min.
24
Next, the blot was rinsed 2 x 3min with deionized water for their subsequent
25
visualization. Additionally, for erasing the stain on the protein, the blot was rinsed 2 x
26
10min with Congo red destaining, and rinsed several times with deionized water.
27
Recipes: Congo red staining stocking solution contained consisted of 1.0g Congo red,
28
80ml methanol, 0.3g NaCl (sodium chloride) in 100ml DW. The working Congo red
29
staining solution contains 1ml stocking solution and 9 ml of 0.1～0.2 M Acetic acid.
30
Congo red destaining: running buffer consisted of 0.025 M Tris, 0.2 M glycine, and 4
1
0.1% SDS. Stained membranes were scanned on Chemiscope 3600 and Huawei cell
2
phone.
3
Molecular modeling analysis
4
2D or 3D diagrams of Congo red-His protein and Ponceau S-His protein
5
interactions are plotted through LigPlot+ and DINC 2.0. The prediction of
6
PDB-format of His protein are through SWISS-MODEL software.
7
Results and discussion
8
Congo red is an organic compound and was first synthesized in 1883 by Paul
9
Böttiger [9]. Seema Mehta et.al 1998 firstly demonstrated that Congo red dye could
10
effectively stain NC membrane and acrylamide [7]. However, the sensitivity of this
11
protocol to visualizing proteins in NC membrane were much lower and approximately
12
500ng. In addition, the authors did not confirm that the specificity of transferred
13
proteins visualized by either Ponceau S or Congo red using Western blots. Therefore,
14
it is necessary to improve this method. Based on the previous method, sensitivity for
15
staining protein in NC and PVDF membranes using Congo red were obviously
16
enhanced when Congo red dye was dissolved in 80% methanol, pH 3.0 (referring to
17
recipes in Methods). During the experiment, we discovered that the same proteins
18
staining results could be acquired between 0.1M and 0.2M Acetic acid in Congo red
19
working solution (data not shown). Further, results of dyeing proteins in 8% methanol
20
of working Congo red staining solution are better than 5% methanol (data not shown).
21
As for the mechanisms involved in the process, there are no reports at present. Thus,
22
we speculate that higher concentration methanol in working staining solution may
23
increase the negative charge on the Congo red and remarkably enhances the binding
24
capability of Congo red to proteins. Besides, we established very effective and readily
25
available materials of Congo red destaining solution (referring to Methods). The stain
26
erasing procedure for NC and PVDF membranes takes just about 10～20min to
27
remove the stain completely from the proteins and does not interfere with downstream
28
Western blots results. Besides, the present destaining method provides a bright
29
uniform background. Interestingly, we observed that the stained protein NC and
30
PVDF membranes could be preserved in 4°C in DW for 1 month without fading. 5
1
Fig.2 shows the sensitivity of Congo red stain with that of Ponceau S stain on
2
proteins bound to nitrocellulose (A, B) and PVDF (C, D) membranes, respectively.
3
Next, Variable amounts of unstained molecular weight standard markers were
4
separated and then transferred to the blots membranes from 5–10% Tris–glycine
5
SDS–PAGE gels. Fig.2 (A) indicate that sensitivity of detection proteins for Congo
6
red is approximately as low as 20ng and for Ponceau S is about 60ng. As shown in
7
Fig.2 (A), the even signal intensity of all markers dark blue-violet bands are still
8
stronger in line 1 of 20ng. Surprisingly, Congo red staining can detect even a faint
9
10ng bands, which are not be visible through Ponceau S in Fig.2 (A-B). Additionally,
10
the whole proteins are more easily bound to nitrocellulose than PVDF membranes,
11
while using Congo red or Ponceau S stain Fig.2 (A-D). Thus, the results apparently
12
suggest that sensitivity of approximately is approximately 3.0-fold greater than that of
13
Ponceau S. Taken together, these data clearly hint that abilities of Congo red protein
14
stain is more sensitive than Ponceau S.
15
Previous studies showed that reversible staining process can be achieved by
16
MemCode kits (the actual composition of the commercial Ponceau S is not provided
17
by the manufacturer) [1], Ponceau S and Amido black 10B [6]. At the same time,
18
Congo red protein stain also has shown the better reversible features in our
19
experiments. After proteins staining and destaining, Fig.3.D and Fig.4.E indicate that
20
the proteins from different sources can be successfully detected by immunoblot
21
analysis on nitrocellulose membrane. These results clearly demonstrate that Congo
22
red stain is compatible with Western blotting and can precisely display whether
23
proteins are successfully electroblotted to nitrocellulose or PVDF membranes in
24
advance. On the other hand, we can draw a conclusion from Fig.3.A-C and Fig.4.A-D
25
that staining results of Congo red are better for NC than PVDF membranes.
26
To further study the possible staining mechanism at protein and Congo red as
27
well as Ponceau S dye, molecular docking research were conducted, and His-NogoA
28
△20 peptide was selected as a representative protein. Prior to docking, His protein
29
structure (amino acid 564-749) was predicted by SWISS-MODEL. In Fig.5, a plot
30
demonstrates that two different dyes bind to His protein. It was obviously observed 6
1
that the binding sites is mainly hydrophobic residue contacts. The five residues,
2
including MET-204, ALA-200, LEU-203, GLU-199 and THR-196, are implicated in
3
hydrophobic contacts of Congo red staining group (Fig.5A). However, only four
4
hydrophobic contacts, such as MET-204, LEU-208, LYS-205, VAL-201, are
5
discovered in Ponceau S dyeing (Fig.5B). Furthermore, low NaCl concentration in
6
working solutions may enhance hydrophobic interaction [11] between His-protein and
7
dye Congo red. On the other hand, the prediction results show that Congo red and
8
Ponceau S may form stable hydrogen bonds with residues GLN-195 or GLN-197 at
9
binding site of His proteins. All in all, the forecasts of the preliminary 2D data reveals
10
possible molecular mechanisms for Congo red staining are better than Ponceau S.
11
In order to clearly display 3D structures under different staining conditions,
12
PyMol software was used in Fig.6. 3D images of Congo red-His protein binding
13
models show that the number of potential hydrogen bonds (Fig.6A) are more than
14
Ponceau S (Fig.6B). The hydrogen bond is stronger than an electrostatic interaction
15
(van der Waals interaction) , but weaker than covalent or ionic bonds [2]. Meanwhile,
16
statistics derived from DINC 2.0 in Fig.7 also confirm that binding energy in Congo
17
red-his protein are relative greater (Fig.7A-B) than Ponceau S (Fig.7C-D). Consistent
18
with previous 2D findings, the preliminary 3D prediction results also confirm that
19
Congo red-his protein interactive stability are better than Ponceau S.
20
Recently review paper showed that Ponceau S is a negatively charged sodium
21
salt of a diazo dye and binds to positively charged amino acids as well as non-polar
22
regions of proteins [8]. Interestingly, Congo red possesses two negatively charged
23
sulphonic acid groups [5]. Therefore, we present a new hypothesis that Congo red
24
could bind to positively charged amino acids residues of His-protein by electrostatic
25
interactions at~pH3.0 solutions. Based on the above analysis, we propose that
26
interaction between His-protein and Congo red or Ponceau S occurs mainly via
27
hydrogen bonding, hydrophobic and electrostatic interactions, which may promote the
28
stability of binding between His-protein and different dyes.
29
In this study, we have developed a modified procedure for fast, high-efficiency
30
and reversible staining of proteins on NC and PVDF membranes using Congo red. 7
1
The specificity and sensitivity of the current approach have been confirmed by
2
western Blot analysis. A likely interactive mechanism between Congo red and
3
proteins also are explored by molecular docking software. Additionally, compared to
4
other stain methods, this procedure takes only several minutes to stain and the cost of
5
used materials is less expensive and more economical (Table.1). Regardless, some
6
fluorescence-based stains are reported for detection of electroblotted proteins on NC
7
and PVDF membranes, however, the methods are time-consuming and high-cost [1].
8
Accordingly, Congo red stain would be a preferable staining method for Western
9
blots.
10 11 12
Figure legends Figure.1. Staining protocol for proteins on blot membranes using Congo red or Ponceau S.
13
Figure.2. Comparison the sensitivities of staining with Congo red and Ponceau S
14
on nitrocellulose and PVDF membranes using unstained Protein Ladder.
15
Nitrocellulose (A, C) and PVDF (B, D) blots. Each of staining steps was described in
16
Methods Section. The amounts of standard unstained proteins marker in each wells
17
are as follows: lane 0, 10 ng; lane 1, 20 ng; lane 2, 60ng; lane 3, 90 ng; lane 4, 120ng;
18
lane 5, 150 ng; lane 6, 180 ng; lane 7, 210 ng; lane 8, Prestained Protein Marker .
19
Blots C and D were stained with 0.1% Ponceau S in 5% acetic acid for 5min and
20
rinsed with DW for 1-2min.
21
Figure.3. Western blot analysis of His protein by chemiluminescent detection
22
after Congo red staining, destaining, and Ponceau S staining on nitrocellulose
23
membrane. Various amounts of His protein were applied onto 5–10% Tris–glycine
24
SDS–PAGE gels and the protein was transferred from the SDS-PAGE gels (1.0mm)
25
onto nitrocellulose membranes (0.45µm). The membrane was subjected to Congo red
26
staining (Blot A), destaining (Blot B), and Ponceau S staining (Blot C) procedures as
27
described under Materials and methods. The nitrocellulose membrane was then
28
detected with anti-His Mouse Monoclonal Antibody (Blot D) followed by
29
chemiluminescent method. Lane 1, Prestained Protein Marker; lane 2, 500ng; lane 3,
30
300 ng; lane 4,150ng; lane 5,100 ng; lane 6, 50ng; lane7, 25ng. 8
1
Figure.4. Western blot analysis of p44/42 MAPK (Erk1/2) by chemiluminescent
2
detection nitrocellulose membrane after Congo red staining, destaining, and Ponceau
3
S staining on nitrocellulose membrane or PVDF membrane.
4
Various amounts of NSCs extract proteins were applied to onto 5–10% Tris–
5
glycine SDS–PAGE gels (1.0mm) and the proteins were electroblotted onto
6
nitrocellulose membrane and PVDF (0.45µm). The membrane was subjected to
7
Congo red staining (A: nitrocellulose membrane and C: PVDF), and Ponceau S
8
staining (B: nitrocellulose membrane and D: PVDF). The nitrocellulose membrane of
9
Figure.4 (A) was detained then incubated with primary p42/p44 ERK antibody and
10
detected using Beyotime Biotechnology chemiluminescent Substrate (Blot E). Lane 1,
11
12µg; lane 2, 8µg; lane 3, 4µg; M, prestained marker proteins.
12
Figure.5. 2D diagrams of Congo red-His protein and Ponceau S-His protein
13
interactions. The plots were generated by LigPlot+ according to the manual
14
instructions. PDB-format of His protein are predicated by SWISS-MODEL. The
15
left-hand (A) plot shows Congo red bound to His protein, while the right-hand (B)
16
plot shows the binding of Ponceau S to His protein. Hydrogen bonds are shown as
17
green dotted lines. Ligand bonds indicated in purple. The red circles and ellipses
18
represent non-ligand protein residues that may be involved in hydrophobic contacts.
19
Figure.6. 3D diagrams of Congo red-His protein and Ponceau S-His protein
20
bindings. The plots were generated by PyMol software. The upper (A; 5 hydrogen
21
bonds) plot shows Congo red. The lower (B; 3 hydrogen bonds) plot shows Ponceau S.
22
The gray α-helix indicate 3D structure of His protein. Hydrogen bonds are shown as
23
yellow dotted lines. The numbers shows length of Hydrogen bonds (unit: Å).
24
Figure.7. Analysis of Congo red-His protein and Ponceau S-His protein binding
25
energy with DINC 2.0. The (A) plot shows Congo red bound to His protein and
26
binding energy statistics (B). The (C) plot shows Ponceau S bound to His protein and
27
binding energy statistics (D).
28 29 30 9
1 2
Acknowledgements
3
LigPlot+ is available from http://www.ebi.ac.uk/thornton-srv/ software/LigPlus.
4
DINC 2.0 is available from http://dinc.kavrakilab.org/ software. SWISS-MODEL is
5
available from https://swissmodel.expasy.org/interactive software.
6
Funding
7
The study was supported by the Zhejiang Provincial Natural Science Foundation of
8
China (grant no, LY18H090013) and Wenzhou Municipal Science & Technology
9
Bureau Fund (grant no, Y20180094).
10
Availability of data and materials
11
All data generated or analyzed during this study are included in this published article.
12
Abbreviations
13
Deionized water: DW
14
Authors’ contributions
15
Wang JL, Zhao-Li, Mei-Qi Li and Xu CJ analyzed the data; Xu CJ, Wei-Guang Chen,
16
and Wang JL wrote the manuscript.
17
Ethics approval and consent to participate
18
Not applicable.
19
Patient consent for publication 10
1
Not applicable.
2
Competing interests
3
The authors declare that they have no competing interests.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Reference [1] B.S. Antharavally, B. Carter, P.A. Bell, A. Krishna Mallia, A high-affinity reversible protein stain for Western blots, Analytical biochemistry, 329 (2004) 276-280. [2] J. Berg, J. Tymoczko, L. Stryer, Chemical bonds in biochemistry, Biochemistry. Fifth edition. WH Freeman, New York. Section, 1 (2002). [3] A. Dsouza, R.H. Scofield, Protein Stains to Detect Antigen on Membranes, Methods in molecular biology (Clifton, N.J.), 1314 (2015) 33-40. [4] A. Goldman, S. Harper, D.W. Speicher, Detection of Proteins on Blot Membranes, Current protocols in protein science, 86 (2016) 10.18.11-10.18.11. [5] W.E. Klunk, J.W. Pettegrew, D.J. Abraham, Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 37 (1989) 1273-1281. [6] B. Magi, L. Bianchi, Immunoblotting of 2-DE Separated Proteins, 2009. [7] S. Mehta, Y.S. Rajput, A method for staining of proteins in nitrocellulose membrane and acrylamide gel using Congo red dye, Analytical biochemistry, 263 (1998) 248-251. [8] C.P. Moritz, Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots, Proteomics, 17 (2017). [9] D.P. Steensma, "Congo" red: out of Africa?, Archives of pathology & laboratory medicine, 125 (2001) 250-252. [10] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proceedings of the National Academy of Sciences of the United States of America, 76 (1979) 4350-4354. [11] K. Tsumoto, D. Ejima, A.M. Senczuk, Y. Kita, T. Arakawa, Effects of salts on protein-surface interactions: applications for column chromatography, Journal of pharmaceutical sciences, 96 (2007) 1677-1690. [12] J.L. Wang, J.J. Wang, Z.N. Cai, C.J. Xu, The effect of curcumin on the differentiation, apoptosis and cell cycle of neural stem cells is mediated through inhibiting autophagy by the modulation of Atg7 and p62, International journal of molecular medicine, 42 (2018) 2481-2488. [13] C.J. Xu, J.L. Wang, P. Jing, L. Min, Tph2 Genetic Ablation Contributes to Senile Plaque Load and Astrogliosis in APP/PS1 Mice, Current Alzheimer research, 16 (2019) 219-232. [14] C.R. Yonan, P.T. Duong, F.N. Chang, High-efficiency staining of proteins on different blot membranes, Analytical biochemistry, 338 (2005) 159-161.
37
11
Highlights 1. We developed a sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes. 2. About 20ng proteins could be detected in 3 min on NC and PVDF membranes by Congo red staining. 3. Congo red staining is much cheaper and more convenient compared to commercial MemCode kit.
The authors declare that they have no competing interests.
S0003-2697(19)31082-6
DOI:
https://doi.org/10.1016/j.ab.2020.113579
Reference:
YABIO 113579
To appear in:
Analytical Biochemistry
Received Date: 1 November 2019 Revised Date:
4 January 2020
Accepted Date: 6 January 2020
Please cite this article as: J.-L. Wang, Z. Li, M.-Q. Li, W.-G. Chen, C.-J. Xu, A sensitive and reversible staining of proteins on blot membranes, Analytical Biochemistry (2020), doi: https://doi.org/10.1016/ j.ab.2020.113579. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
1
A sensitive and reversible staining of proteins on blot membranes
2
Jun-Ling Wang1, Li-Zhao2, Mei-Qi Li2, Wei-Guang Chen3#, Chao-Jin Xu3#
3
1
4
University, Wenzhou, Zhejiang 325000; School of 2nd clinical medical sciences,
5
Wenzhou Medical University, Wenzhou, Zhejiang 325035; 3Department of Histology
6
& Embryology, School of Basic Medical Science, Wenzhou Medical University,
7
Wenzhou, Zhejiang 325035, P.R. China
8
#
9
Correspondence to: Dr Chao-Jin Xu, Department of Histology & Embryology, School
10
of Basic Medical Science, Wenzhou Medical University, Cha Shan university town,
11
No.1 Central North Road, Zhejiang 325035, P.R. China
12
Chao-Jin Xu E-mail: [email protected]
13
Wei-Guang Chen E-mail: [email protected]
14
Wang Jun-Ling Email: [email protected]
15
Li-Zhao Email: [email protected]
16
Mei-Qi Li Email: [email protected]
17
Number of Pages: 11
18
Number of Figures: 7
19
Number of Tables: 1
Center for Reproductive Medicine, Affiliated Hospital 1 of Wenzhou Medical
Co- Correspondence author
20 21 22 23 24 25 26 27 28 29 1
1
Abstract
2
A modified, sensitive and reversible method for protein staining on nitrocellulose
3
(NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western
4
blotting. The method employed Congo red staining to visualize proteins on different
5
blot membranes. Staining of proteins with Congo red dye is more faster procedures.
6
According to the experimental results, approximate 20ng proteins could be detected in
7
3 min in room temperature. The staining on the proteins is easily reversible with
8
Congo red destaining solution for NC and PVDF membranes, so that the blot
9
membranes can be reused for Western blotting. In addition, we confirmed that the
10
staining method is fully compatible with Western blot detection. NC and PVDF
11
membranes treatment with Congo red staining does not interfere with conventional
12
chemiluminescent substrates of peroxidase. As compared to MemCode reversible
13
protein stain kits from Pirece Biotechnology, the staining technique is more sensitive,
14
lower of cost, convenient and not adversely affecting subsequent Western blotting
15
results. On the other hand, the stain is more sensitive than the Ponceau S staining.
16
Therefore, Congo red staining is a promising and ideal alternative for current protein
17
stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated
18
using various 2D and 3D molecular docking and demonstrated potential molecular
19
basis for sensitivity of Congo red staining are higher than Ponceau S.
20 21
Key words: Protein stain; Congo red; Nitrocellulose and PVDF membranes; Western
22
blotting
23 24
Introduction
25
The western blot (sometimes called the protein immunoblot) is a widely used
26
analytical method for visualizing proteins transferred from gels to nitrocellulose,
27
PVDF (polyvinylidenedifluorid) membranes or other immobilized matrices. Although
28
abundant data are available about protein staining by using of different staining agents
29
on the level of polyacrylamide gels, much less is known on the level of blotting
30
membranes [8]. Towbin et al. firstly described the technique of detecting proteins on 2
1
blot membranes [10]. To stain proteins bound to blot membranes, a series of organic
2
dyes have been developed, such as amido black, Ponceau S, Coomassie blue, colloidal
3
silver/gold, India ink, ferridye, Congo red , MemCode and fluorescent dye, SYPRO
4
Ruby [1, 4]. To my knowledge, of proteins staining, Ponceau S, MemCode, and
5
SYPRO Ruby are directly compatible with subsequent immunoblotting [1, 4]. But
6
some stains, such as Coomassie blue, India ink and amido black, are the least
7
sensitive and not well support compatibility [3, 4, 14]. In addition, it is necessary for
8
an ideal protein stain would be high-specific affinity for proteins and no affinity for
9
other biological material. The dye reagents also should bound to proteins strongly and
10
rapidly under normal application conditions, as well as are commercially available.
11
Further, the stains should be compatible with a variety of blot membranes [1]. It is,
12
therefore, our aims was to develop a highly sensitive and less expensive protein
13
staining method toward all blot membranes. During the process of exploration and
14
based on previously research [7], we unexpectedly discovered that Congo red (Fig.1)
15
staining proteins on NC and PVDF membranes quickly with a sensitivity of detection
16
(about 20ng) comparable to that of Ponceau S. The method is simple and can
17
visualize the protein bands on the blot membranes within 3min in the staining
18
working solution. Staining of transferred proteins (～20ng/band) appear as dark
19
blue-violet bands on a white background after rinsing with DW. Besides, removing
20
the dyes from the blot takes about two times at 10minute intervals in destaining
21
solution for subsequent immunological detection. Additionally, sensitivity of detecting
22
proteins on blotting membranes can be up to around 25-fold than previous Congo red
23
staining protocol.
24
All in all, the stain is more sensitive than the Ponceau S stain and much cheaper
25
than MemCode with the properties of an ideal stain. Consequently, we conclude that
26
Congo red staining should be the preferred reversible staining after proteins transfer in
27
future Western blot approaches.
28
Materials and methods
29
Materials
30
PageRuler™ Unstained Protein Ladder was from Thermo Fisher Scientific 3
1
Biotechnology. Dual Color Prestained Protein Marker was from Epizyme
2
Biotechnology. Congo red ( Sigma-Aldrich); His-Tag Mouse Monoclonal Antibody
3
(Affinity
4
Biotechnology); p44/42 MAPK (Erk1/2) rabbit mAb (Cell Signaling Technology);
5
HRP-conjugated goat anti-rabbit IgG (Beyotime Biotechnology). Ponceau 3R were
6
from Sigma-Aldrich. Nitrocellulose membrane (NC) and Polyvinylidenefluoride
7
(PVDF) and BeyoECL Star chemiluminescent substrate were from Beyotime
8
Biotechnology. Methanol (Changshu Hongsheng Fine Chemical Co., Ltd; China).
9
Neural stem cells (NSCs) were prepared from embryonic day 17 (E17) SD rats
10
(supplied by Chinese Academy of Sciences) according to the protocols established by
11
our group [12].
12
Western blot analysis
Biosciences);
HRP-conjugated
goat
anti-mouse
IgG
(Beyotime
13
Thermo Scientific Unstained Protein Molecular Weight Marker containing
14
several native proteins (size from 10 to 200 kDa), NSCs lysate proteins, and purified
15
His-NogoA△20 protein (amino acid peptide 564-749,~23KD; unpublished data)
16
were used as model proteins. Proteins were separated on 5~10% Tris–glycine SDS–
17
PAGE gels and transferred onto nitrocellulose or PVDF membranes, according to the
18
methods established by us [12, 13]. The total protein concentration was measured by
19
Detergent Compatible Bradford Protein Assay Kit (Beyotime Biotechnology). Images
20
were acquired on the Clinx imager (www.clinx.cn).
21
Staining, destaining, and stain erasing procedure for NC and PVDF membranes
22
After electro blotting, the NC and PVDF membrane were rinsed with deionized
23
water (DW) for 2 x 5min, and stained with Congo red staining solution for 3 min.
24
Next, the blot was rinsed 2 x 3min with deionized water for their subsequent
25
visualization. Additionally, for erasing the stain on the protein, the blot was rinsed 2 x
26
10min with Congo red destaining, and rinsed several times with deionized water.
27
Recipes: Congo red staining stocking solution contained consisted of 1.0g Congo red,
28
80ml methanol, 0.3g NaCl (sodium chloride) in 100ml DW. The working Congo red
29
staining solution contains 1ml stocking solution and 9 ml of 0.1～0.2 M Acetic acid.
30
Congo red destaining: running buffer consisted of 0.025 M Tris, 0.2 M glycine, and 4
1
0.1% SDS. Stained membranes were scanned on Chemiscope 3600 and Huawei cell
2
phone.
3
Molecular modeling analysis
4
2D or 3D diagrams of Congo red-His protein and Ponceau S-His protein
5
interactions are plotted through LigPlot+ and DINC 2.0. The prediction of
6
PDB-format of His protein are through SWISS-MODEL software.
7
Results and discussion
8
Congo red is an organic compound and was first synthesized in 1883 by Paul
9
Böttiger [9]. Seema Mehta et.al 1998 firstly demonstrated that Congo red dye could
10
effectively stain NC membrane and acrylamide [7]. However, the sensitivity of this
11
protocol to visualizing proteins in NC membrane were much lower and approximately
12
500ng. In addition, the authors did not confirm that the specificity of transferred
13
proteins visualized by either Ponceau S or Congo red using Western blots. Therefore,
14
it is necessary to improve this method. Based on the previous method, sensitivity for
15
staining protein in NC and PVDF membranes using Congo red were obviously
16
enhanced when Congo red dye was dissolved in 80% methanol, pH 3.0 (referring to
17
recipes in Methods). During the experiment, we discovered that the same proteins
18
staining results could be acquired between 0.1M and 0.2M Acetic acid in Congo red
19
working solution (data not shown). Further, results of dyeing proteins in 8% methanol
20
of working Congo red staining solution are better than 5% methanol (data not shown).
21
As for the mechanisms involved in the process, there are no reports at present. Thus,
22
we speculate that higher concentration methanol in working staining solution may
23
increase the negative charge on the Congo red and remarkably enhances the binding
24
capability of Congo red to proteins. Besides, we established very effective and readily
25
available materials of Congo red destaining solution (referring to Methods). The stain
26
erasing procedure for NC and PVDF membranes takes just about 10～20min to
27
remove the stain completely from the proteins and does not interfere with downstream
28
Western blots results. Besides, the present destaining method provides a bright
29
uniform background. Interestingly, we observed that the stained protein NC and
30
PVDF membranes could be preserved in 4°C in DW for 1 month without fading. 5
1
Fig.2 shows the sensitivity of Congo red stain with that of Ponceau S stain on
2
proteins bound to nitrocellulose (A, B) and PVDF (C, D) membranes, respectively.
3
Next, Variable amounts of unstained molecular weight standard markers were
4
separated and then transferred to the blots membranes from 5–10% Tris–glycine
5
SDS–PAGE gels. Fig.2 (A) indicate that sensitivity of detection proteins for Congo
6
red is approximately as low as 20ng and for Ponceau S is about 60ng. As shown in
7
Fig.2 (A), the even signal intensity of all markers dark blue-violet bands are still
8
stronger in line 1 of 20ng. Surprisingly, Congo red staining can detect even a faint
9
10ng bands, which are not be visible through Ponceau S in Fig.2 (A-B). Additionally,
10
the whole proteins are more easily bound to nitrocellulose than PVDF membranes,
11
while using Congo red or Ponceau S stain Fig.2 (A-D). Thus, the results apparently
12
suggest that sensitivity of approximately is approximately 3.0-fold greater than that of
13
Ponceau S. Taken together, these data clearly hint that abilities of Congo red protein
14
stain is more sensitive than Ponceau S.
15
Previous studies showed that reversible staining process can be achieved by
16
MemCode kits (the actual composition of the commercial Ponceau S is not provided
17
by the manufacturer) [1], Ponceau S and Amido black 10B [6]. At the same time,
18
Congo red protein stain also has shown the better reversible features in our
19
experiments. After proteins staining and destaining, Fig.3.D and Fig.4.E indicate that
20
the proteins from different sources can be successfully detected by immunoblot
21
analysis on nitrocellulose membrane. These results clearly demonstrate that Congo
22
red stain is compatible with Western blotting and can precisely display whether
23
proteins are successfully electroblotted to nitrocellulose or PVDF membranes in
24
advance. On the other hand, we can draw a conclusion from Fig.3.A-C and Fig.4.A-D
25
that staining results of Congo red are better for NC than PVDF membranes.
26
To further study the possible staining mechanism at protein and Congo red as
27
well as Ponceau S dye, molecular docking research were conducted, and His-NogoA
28
△20 peptide was selected as a representative protein. Prior to docking, His protein
29
structure (amino acid 564-749) was predicted by SWISS-MODEL. In Fig.5, a plot
30
demonstrates that two different dyes bind to His protein. It was obviously observed 6
1
that the binding sites is mainly hydrophobic residue contacts. The five residues,
2
including MET-204, ALA-200, LEU-203, GLU-199 and THR-196, are implicated in
3
hydrophobic contacts of Congo red staining group (Fig.5A). However, only four
4
hydrophobic contacts, such as MET-204, LEU-208, LYS-205, VAL-201, are
5
discovered in Ponceau S dyeing (Fig.5B). Furthermore, low NaCl concentration in
6
working solutions may enhance hydrophobic interaction [11] between His-protein and
7
dye Congo red. On the other hand, the prediction results show that Congo red and
8
Ponceau S may form stable hydrogen bonds with residues GLN-195 or GLN-197 at
9
binding site of His proteins. All in all, the forecasts of the preliminary 2D data reveals
10
possible molecular mechanisms for Congo red staining are better than Ponceau S.
11
In order to clearly display 3D structures under different staining conditions,
12
PyMol software was used in Fig.6. 3D images of Congo red-His protein binding
13
models show that the number of potential hydrogen bonds (Fig.6A) are more than
14
Ponceau S (Fig.6B). The hydrogen bond is stronger than an electrostatic interaction
15
(van der Waals interaction) , but weaker than covalent or ionic bonds [2]. Meanwhile,
16
statistics derived from DINC 2.0 in Fig.7 also confirm that binding energy in Congo
17
red-his protein are relative greater (Fig.7A-B) than Ponceau S (Fig.7C-D). Consistent
18
with previous 2D findings, the preliminary 3D prediction results also confirm that
19
Congo red-his protein interactive stability are better than Ponceau S.
20
Recently review paper showed that Ponceau S is a negatively charged sodium
21
salt of a diazo dye and binds to positively charged amino acids as well as non-polar
22
regions of proteins [8]. Interestingly, Congo red possesses two negatively charged
23
sulphonic acid groups [5]. Therefore, we present a new hypothesis that Congo red
24
could bind to positively charged amino acids residues of His-protein by electrostatic
25
interactions at~pH3.0 solutions. Based on the above analysis, we propose that
26
interaction between His-protein and Congo red or Ponceau S occurs mainly via
27
hydrogen bonding, hydrophobic and electrostatic interactions, which may promote the
28
stability of binding between His-protein and different dyes.
29
In this study, we have developed a modified procedure for fast, high-efficiency
30
and reversible staining of proteins on NC and PVDF membranes using Congo red. 7
1
The specificity and sensitivity of the current approach have been confirmed by
2
western Blot analysis. A likely interactive mechanism between Congo red and
3
proteins also are explored by molecular docking software. Additionally, compared to
4
other stain methods, this procedure takes only several minutes to stain and the cost of
5
used materials is less expensive and more economical (Table.1). Regardless, some
6
fluorescence-based stains are reported for detection of electroblotted proteins on NC
7
and PVDF membranes, however, the methods are time-consuming and high-cost [1].
8
Accordingly, Congo red stain would be a preferable staining method for Western
9
blots.
10 11 12
Figure legends Figure.1. Staining protocol for proteins on blot membranes using Congo red or Ponceau S.
13
Figure.2. Comparison the sensitivities of staining with Congo red and Ponceau S
14
on nitrocellulose and PVDF membranes using unstained Protein Ladder.
15
Nitrocellulose (A, C) and PVDF (B, D) blots. Each of staining steps was described in
16
Methods Section. The amounts of standard unstained proteins marker in each wells
17
are as follows: lane 0, 10 ng; lane 1, 20 ng; lane 2, 60ng; lane 3, 90 ng; lane 4, 120ng;
18
lane 5, 150 ng; lane 6, 180 ng; lane 7, 210 ng; lane 8, Prestained Protein Marker .
19
Blots C and D were stained with 0.1% Ponceau S in 5% acetic acid for 5min and
20
rinsed with DW for 1-2min.
21
Figure.3. Western blot analysis of His protein by chemiluminescent detection
22
after Congo red staining, destaining, and Ponceau S staining on nitrocellulose
23
membrane. Various amounts of His protein were applied onto 5–10% Tris–glycine
24
SDS–PAGE gels and the protein was transferred from the SDS-PAGE gels (1.0mm)
25
onto nitrocellulose membranes (0.45µm). The membrane was subjected to Congo red
26
staining (Blot A), destaining (Blot B), and Ponceau S staining (Blot C) procedures as
27
described under Materials and methods. The nitrocellulose membrane was then
28
detected with anti-His Mouse Monoclonal Antibody (Blot D) followed by
29
chemiluminescent method. Lane 1, Prestained Protein Marker; lane 2, 500ng; lane 3,
30
300 ng; lane 4,150ng; lane 5,100 ng; lane 6, 50ng; lane7, 25ng. 8
1
Figure.4. Western blot analysis of p44/42 MAPK (Erk1/2) by chemiluminescent
2
detection nitrocellulose membrane after Congo red staining, destaining, and Ponceau
3
S staining on nitrocellulose membrane or PVDF membrane.
4
Various amounts of NSCs extract proteins were applied to onto 5–10% Tris–
5
glycine SDS–PAGE gels (1.0mm) and the proteins were electroblotted onto
6
nitrocellulose membrane and PVDF (0.45µm). The membrane was subjected to
7
Congo red staining (A: nitrocellulose membrane and C: PVDF), and Ponceau S
8
staining (B: nitrocellulose membrane and D: PVDF). The nitrocellulose membrane of
9
Figure.4 (A) was detained then incubated with primary p42/p44 ERK antibody and
10
detected using Beyotime Biotechnology chemiluminescent Substrate (Blot E). Lane 1,
11
12µg; lane 2, 8µg; lane 3, 4µg; M, prestained marker proteins.
12
Figure.5. 2D diagrams of Congo red-His protein and Ponceau S-His protein
13
interactions. The plots were generated by LigPlot+ according to the manual
14
instructions. PDB-format of His protein are predicated by SWISS-MODEL. The
15
left-hand (A) plot shows Congo red bound to His protein, while the right-hand (B)
16
plot shows the binding of Ponceau S to His protein. Hydrogen bonds are shown as
17
green dotted lines. Ligand bonds indicated in purple. The red circles and ellipses
18
represent non-ligand protein residues that may be involved in hydrophobic contacts.
19
Figure.6. 3D diagrams of Congo red-His protein and Ponceau S-His protein
20
bindings. The plots were generated by PyMol software. The upper (A; 5 hydrogen
21
bonds) plot shows Congo red. The lower (B; 3 hydrogen bonds) plot shows Ponceau S.
22
The gray α-helix indicate 3D structure of His protein. Hydrogen bonds are shown as
23
yellow dotted lines. The numbers shows length of Hydrogen bonds (unit: Å).
24
Figure.7. Analysis of Congo red-His protein and Ponceau S-His protein binding
25
energy with DINC 2.0. The (A) plot shows Congo red bound to His protein and
26
binding energy statistics (B). The (C) plot shows Ponceau S bound to His protein and
27
binding energy statistics (D).
28 29 30 9
1 2
Acknowledgements
3
LigPlot+ is available from http://www.ebi.ac.uk/thornton-srv/ software/LigPlus.
4
DINC 2.0 is available from http://dinc.kavrakilab.org/ software. SWISS-MODEL is
5
available from https://swissmodel.expasy.org/interactive software.
6
Funding
7
The study was supported by the Zhejiang Provincial Natural Science Foundation of
8
China (grant no, LY18H090013) and Wenzhou Municipal Science & Technology
9
Bureau Fund (grant no, Y20180094).
10
Availability of data and materials
11
All data generated or analyzed during this study are included in this published article.
12
Abbreviations
13
Deionized water: DW
14
Authors’ contributions
15
Wang JL, Zhao-Li, Mei-Qi Li and Xu CJ analyzed the data; Xu CJ, Wei-Guang Chen,
16
and Wang JL wrote the manuscript.
17
Ethics approval and consent to participate
18
Not applicable.
19
Patient consent for publication 10
1
Not applicable.
2
Competing interests
3
The authors declare that they have no competing interests.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
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37
11
Highlights 1. We developed a sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes. 2. About 20ng proteins could be detected in 3 min on NC and PVDF membranes by Congo red staining. 3. Congo red staining is much cheaper and more convenient compared to commercial MemCode kit.
The authors declare that they have no competing interests.