Name: fluorescent silver staining of proteins in polyacrylamide gels
Uploaded: 2019-04-21T13:00:00
Description: Watch this Scientific Journal Video about Fluorescent Silver Staining of Proteins in Polyacrylamide Gels at JoVE.com

The authors would like to thank Patrick Chan at the Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, for his technical support. S. X. is grateful for the support from the Swedish Research Council (Grant No. 2017-06344).

1. Preparation of the Gel
Note: The demonstration follows a standard protocol to prepare the gel for staining shortly after SDS-PAGE12. In brief, the following steps describe the preparation of the samples and gel electrophoresis.
<ol>
	<li>Perform SDS-PAGE with 4% - 12% Bis-Tris protein gels (1 mm, 15-well) using a mini gel tank filled with 2-(N-morpholino)ethanesulfonic acid (MES) buffer.</li>
	<li>Dilute the samples with a mixture of distilled water, lithi...

<ol>
	<li>Chevalier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highlights+on+the+capacities+of+&amp;#34;Gel-based&amp;#34;+proteomics.">Highlights on the capacities of &amp;#34;Gel-based&amp;#34; proteomics.</a> Proteome Science. 8 (1), (2010).</li><li>Harris, L. R., Churchward, M. A., Butt, R. H., Coorssen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+detection+methods+for+gel-based+proteomic+analyses.">Assessing detection methods for gel-based proteomic analyses.</a> Journal of Proteome Research. 6 (4), 1418-1425 (2007).</li><li>Gauci, V. J., Wright, E. P., Coorssen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics:+assessing+the+spectrum+of+in-gel+protein+detection+methods.">Quantitative proteomics: assessing the spectrum of in-gel protein detection methods.</a> Journal of Chemical Biology. 4 (1), 3-29 (2011).</li><li>Candiano, G., Bruschi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25 (9), 1327-1333 (2004).</li><li>Chevallet, M., Luche, S., Rabilloud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+proteins+in+polyacrylamide+gels.">Silver staining of proteins in polyacrylamide gels.</a> Nature Protocols. 1 (4), 1852-1858 (2006).</li><li>Jin, L. T., Hwang, S. Y., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+silver+staining+of+protein+in+sodium+dodecyl+sulfate-polyacrylamide+gels+using+an+azo+dye,+calconcarboxylic+acid,+as+a+silver-ion+sensitizer.">Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer.</a> Electrophoresis. 25 (15), 2494-2500 (2004).</li><li>Celis, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A Laboratory Handbook. , (2006).</li><li>Bartsch, H., Arndt, C., Koristka, S., Cartellieri, M., Bachmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+Staining+Techniques+of+Polyacrylamide+Gels.">Silver Staining Techniques of Polyacrylamide Gels.</a> Methods in Molecular Biology. 869 (1), 481-486 (2012).</li><li>Rabilloud, T., Vuillard, L., Gilly, C., Lawrence, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver-staining+of+proteins+in+polyacrylamide+gels:+a+general+overview.">Silver-staining of proteins in polyacrylamide gels: a general overview.</a> Cellular and Molecular Biology. 40 (1), 57-75 (1994).</li><li>Zhou, M., Yu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+by+Two-Dimensional+Polyacrylamide+Gel+Electrophoresis.">Proteomic Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis.</a> Advances in Protein Chemistry. 65 (1), 57-84 (2003).</li><li>Xie, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+Ag+-Tetrazolate+Aggregation+Enables+Efficient+Fluorescent+Biological+Silver+Staining.">Fluorogenic Ag+-Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining.</a> Angewandte Chemie International Edition. 57 (20), 5750-5753 (2018).</li><li>. Protein gel electrophoresis technical handbook Available from: <a target="_blank" href="https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf">https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf</a> (2015)</li></ol>

Presented here is a novel fluorescent silver staining method for proteins in polyacrylamide gels. This strategy integrates conventional silver stains and fluorescent stains. The staining exploits the selective binding of silver ion to proteins as in other silver stains but employs a highly sensitive fluorogenic silver probe TPE-4TA to light up the silver bound proteins. Since the fluorogenic probe TPE-4TA can sense silver ions at a fairly low concentration in the nanomolar range<sup clas...

Many staining methods have been used to visualize proteins following gel electrophoresis, for example using colorimetric dyes such as Coomassie Brilliant Blue, silver stain, fluorescence, or radioactive labeling1,2,3,4. Silver staining is considered to be one of the most sensitive techniques for protein detection requiring simple and cheap reagents. It can be categorized into two major families: the ammoniacal silver stain and the silver nitrate stain5,6. In the alkaline ammoniacal silver method, the silver-diamine complex is produced with ammonia and sodium hydroxide and reduced to metallic silver during development using an acidic formaldehyde solution. The stain accommodates efficiently for basic proteins but shows a compromised performance for acidic and neutral proteins and is, furthermore, limited to classical glycine and taurine electrophoretic systems. In comparison, the silver nitrate stains exploit the high bio-affinity of silver ions to protein, primarily the sulfhydryl and carboxyl groups from the side chains, and tend to stain acidic proteins more efficiently7. After silver ion binding, a developing solution (typically made of a metal carbonate solution containing formaldehyde and sodium thiosulphate) is applied to reduce silver ions to metallic silver grains, which build up a brown-to-dark color to visualize the protein bands.
Although silver staining has been well known for its versatility and high sensitivity since its development in the 1970s8, the method is frequently regarded as tricky. Silver-staining methods have time-restricted steps and show low reproducibility. Since the color of silver stain is usually not uniform and dependent on the reduction step, which is hard to control, the silver stain is not a quantitative method and, thus, not recommended for gel comparison study and protein quantification9. Methods optimized in sensitivity may utilize aldehydes which can also provide a more uniform staining10. However, this is at the expense of further downstream analysis due to the crosslinking of proteins by aldehydes. Fast protocols mostly combine or shorten steps to reduce time, compromising the reproducibility and uniformity of the stain5. As a result, there are numerous silver staining variants within protein gel staining, each optimized to suit certain requirements; for example, simplicity, sensitivity, or peptide recovery rate for downstream analysis. These attributes may also have an impact on each other, and satisfying all requirements in one protocol can be difficult.
In this work, we introduce a new fluorescent silver staining method for protein detection in polyacrylamide gel. In this method, we use a fluorogenic probe for silver ions, TPE-4TA (Figure 1), to visualize the silver-impregnated proteins11. TPE-4TA is designed by the aggregation-induced emission (AIE) principle. It is non-emissive when dissolved in aqueous solution, but is highly emissive in the presence of silver ions. By replacing the chromogenic development in traditional silver stains with a fluorogenic developing step, the fluorescent silver method enables the robust staining of total proteins with a reduced background.
Furthermore, the fluorescent silver stain showed a good dynamic linear range for protein quantification, which is comparable with the widely-used SYPRO Ruby stain and not achievable with traditional silver stains. The gel can be imaged on commonly used gel documentation systems with an ultraviolet lamp (excitation wavelength: 302/365 nm channel; emission: ~490 - 530 nm) at many biological labs.

<table>
	<tbody>
		<tr>
			<td> 
			LDS Sample Buffer (4X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0007</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td> 
			4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0323BOX</td>
			<td>Precast gel</td>
		</tr>
		<tr>
			<td>Sample Reducing Agent (10X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0004</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0002</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Mini Gel Tank</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25977</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>300W Power Supply (230 VAC)</td>
			<td>Thermo Fisher Scientific</td>
			<td>PS0301</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Unstained Protein Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26614</td>
			<td>Sample</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma-Aldrich</td>
			<td>31630-25G-R</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Bragg and co.</td>
			<td>42520J</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>J.T. Baker</td>
			<td>103201A</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Milli-Q Synthesis A10</td>
			<td>Merk</td>
			<td>-</td>
			<td>Provides 18.2 M&#937;.cm water</td>
		</tr>
		<tr>
			<td>gel documentation system (c600 model)</td>
			<td>Azure biosystems</td>
			<td>-</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

fluorescent silver staining of proteins in polyacrylamide gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

The protein bands stained by the fluorescent silver stain exhibit an intense green fluorescence under a 365 nm UV lamp. All 14 protein bands (10 - 200 kDa), from top to bottom, were clearly visible, correlating well with the 14 red-colored ones stained by the SYPRO Ruby dye (Figure 2)10.
Regarding quantitative protein detection, the gels were imaged by a gel imaging system us...

Watch this Scientific Journal Video about Fluorescent Silver Staining of Proteins in Polyacrylamide Gels at JoVE.com

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

This is a novel gel-staining method that utilizes a fluorogenic silver probe to convert the traditional silver staining into a fluorescent silver staining. This method showed improved performance compared with conventional silver nitrate staining and SYPRO Ruby staining while using a simple and straightforward protocol without aldehydes. Demonstrating the procedure will be Alex Wong, a research assistant from my laboratory.To begin this procedure, dilute the protein samples with a solution containing distilled water, LDS buffer, and a sample-reducing agent. Set up a mini gel tank filled with MES buffer to perform SDS-PAGE with a 4-12%Bis-Tris protein gel. Load the wells with the prepared protein sample, then run the gel at a constant voltage of 200 volts for 30 minutes.After electrophoresis, submerge the gel in a 100-milliliter solution containing ethanol and acetic acid on an orbital shaker and incubate twice for 30 minutes each at room temperature while shaking at 50 rpm. Next, wash the gel three times with ultrapure water in a clean container with each wash lasting 10 minutes. First, dissolve 0.01 grams of silver nitrate in 10 milliliters of ultrapure water to prepare a stock solution with a concentration of 0.1%Add 100 microliters of the stock solution into 100 milliliters of ultrapure water to make the silver nitrate working solution.In a fume hood, submerge the gel in 100 milliliters of the silver nitrate working solution in a sealed glass chamber. Use aluminum foil to protect the gel from light during impregnation and incubate for one hour while shaking at 50 rpm on an orbital shaker. After this, wash the gel twice with ultrapure water in a clean container, with each wash using approximately 100 milliliters of water and lasting 60 seconds.It is important to use ultrapure water to clean the gel after the silver impregnation step to minimize background staining. First, add 50 milliliters of ultrapure water to three milligrams of TPE-4TA dye. Sonicate the solution for three minutes and add five microliters of one-molar sodium hydroxide solution in between each sonication session to help dissolve the dye, usually up to three times.Then check the fluorescence of the solution under a 365-nanometer UV lamp to ensure that the dye is fully dissolved. Only weak or non-emissive solutions indicate full dissolution. To prepare the fluorogenic developing solution, add 10 milliliters of the TPE-4TA stock solution to 90 milliliters of ultrapure water.Use a pH meter to check the pH of the solution. Tune the solution to a pH between 7 and 9, using diluted sodium hydroxide solution or acetic acid if the pH is out of range. Next transfer the gel to a clean and sealable container with 100 milliliters of the fluorogenic developing solution and ensure that the gel is completely immersed.Seal the container and cover it to protect it from light. Shake the container overnight on an orbital shaker at 50 rpm and at room temperature. Transfer the gel to a clean container and destain it in 100 milliliters of 10%ethanol for 30 minutes.Then rinse the gel in ultrapure water for five minutes. The gel can be visualized on a benchtop transilluminator or imaged on a gel documentation machine at the 365-nanometer channel or the 302-nanometer channel. In this study, a novel fluorescent silver staining method is used to stain proteins in polyacrylamide gels.The protein bands stained by the fluorescent silver stain exhibit an intense green under a 365-nanometer UV lamp. All 14 protein bands are clearly visible and correlate well with the red-colored bands stained by the SYPRO Ruby dye. This fluorescent silver staining method appears to have a high resolution.For some protein bands, the sensitivity of the fluorescent silver stain is also slightly better than that of the fluorescent SYPRO Ruby stain. In particular the performance of this stain is improved for the protein bands between 10 and 40 kilodaltons, which indicates that the new method is particularly useful for the detection of proteins with a low molecular weight. As can be seen, the data also suggests that the fluorescent silver stain gives a good and uniform linearity for all 14 proteins over a relatively broad range of protein quantification.While the silver nitrate stain gave a high level of background signal and distorted peaks, the fluorescent silver stain detected the bands with good contrast and uniform intensity distribution comparable to the SYPRO Ruby stain across all 14 proteins. Washing steps are important as they limit the residual fixation solution from disrupting silver impregnation or fluorogenic development. When using silver nitrate, avoid skin contact.Clean any spillages immediately. Wear protective clothing and gloves and dispose as chemical waste.

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