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, lithium dodecyl sulfate (LDS) buffer, and a sample-reducing agent.</li>
	<li>Load the first lane with double the amount of stock (10 &#181;L), followed by the normal stock amount (5 &#181;L) and a series of twofold dilutions of the stock thereafter (13 dilutions, from 2x to 8192x).</li>
	<li>Run the gel at a constant voltage of 200 V for 30 min.</li>
</ol>
2. Fixation of the Gel
<ol>
	<li>After electrophoresis, submerge the gels in a 100 mL solution of 40% ethanol/10% acetic acid on an orbital shaker at 50 rpm at room temperature 2x (each for 30 min), or overnight at 4 &#176;C.</li>
	<li>Wash the gels 3 x 10 min each in ultrapure water in a clean container. The washing step is critical. If the acid from the fixing solution is not removed properly in the fluorogenic developing step, the excessive TPE-4TA will be activated by the acid and lead to strong background fluorescence in the gel.</li>
</ol>
3. Preparation of the AgNO3 Solution and Silver Impregnation of the Gel 
<ol>
	<li>To make the AgNO3 solution (0.0001%) for the impregnation, first, dissolve 0.01 g of AgNO3 in 10 mL of ultrapure water to prepare a 0.1% AgNO3 stock solution. Next, add 100 &#181;L of the 0.1% AgNO3 stock solution into 100 mL of ultrapure water to make the working solution. 
	Note: The AgNO3 solution should be stored in the dark before use.</li>
	<li>Impregnate the gel with 100 mL of silver working solution for 1 h on an orbital shaker at 50 rpm in a sealed glass container. It is critical to perform the silver impregnation under a fume hood, protected from light with aluminum foil.</li>
	<li>Wash the gel with ultrapure water (about 100 mL) in a clean container for 2 x 60 s.</li>
</ol>
4. Fluorogenic Development of the Gel
<ol>
	<li>To prepare the dye stock solution (0.1 mM), add 3.0 mg of the TPE-4TA dye in 50 mL of ultrapure water. Sonicate the solution for a few minutes and add some NaOH solution (for example 1 M) to help dissolve the dye.</li>
	<li>Check the fluorescence of the solution under a 365 nm UV lamp to ensure that the dye molecule is fully dissolved. Only weak or nonemissive solutions indicate full dissolution. 
	Note: The dye TPE-4TA was synthesized following the protocol recently reported by Xie et al.10 The TPE-4TA solution is very stable and can be kept in the dark for months.</li>
	<li>To prepare 100 mL of the fluorogenic developing solution (10 &#181;M), add 10 mL of the TPE-4TA stock solution into 90 mL of ultrapure water. Check the pH of the solution using a pH meter and tune it to 7 - 9 using a sodium hydroxide solution (1 &#181;M).</li>
	<li>Transfer the gel to a clean and sealable container with 100 mL of the fluorogenic developing solution. Make sure the gel is totally immersed in the solution. Seal the container. Cover it from light and shake it overnight on an orbital shaker at 50 rpm at room temperature. Alternatively, the incubation step can also be shortened to about ~2 h by preheating the AIE developing solution to 80 &#176;C for staining and then leaving it at room temperature.</li>
</ol>
5. Destaining and Imaging
<ol>
	<li>Transfer the gel to a clean container and destain it in 100 mL of 10% ethanol for 30 min. 
	Note: Water alone can be used to wash the gel. However, it is more time-efficient to use a 10% ethanol solution for the destaining. This will help to reduce the destaining process from hours to 30 min.</li>
	<li>Rinse the gel in ultrapure water for 5 min.</li>
	<li>Image the gel at the 365 nm channel or 302 nm channel by a gel documentation machine.</li>
</ol>

<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>

A patent application on this fluorescent silver staining has been filed.

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>&lt;br/&gt; LDS Sample Buffer (4X)</td><td>Thermo Fisher Scientific</td><td>NP0007</td><td>Reagent</td></tr><tr><td>&lt;br/&gt; 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&amp;Omega;.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 ...

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Biochemistry

14.3K Views.  Karolinska Institutet. Dit is een nieuwe gel-kleuring methode die gebruik maakt van een fluorogenic zilveren sonde om de traditionele zilveren kleuring om te zetten in een fluorescerende zilveren kleuring. Deze methode toonde verbeterde prestaties in vergelijking met conventionele zilvernitraat vlekken en SYPRO Ruby vlekken tijdens het gebruik van een eenvoudig en eenvoudig protocol zonder aldehyden. Het aantonen van de procedure zal Alex Wong zijn, een onderzoeksassistent van mijn laboratorium.Om met deze p...

Video: Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Genetics

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Biology

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Fluorescent Silver Staining: A Technique to Visualize Total Protein in Polyacrylamide Gels

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 this 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 1 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 3 milligrams of TPE-4TA dye. Sonicate the solution for 3 minutes, and add 5 microliters of 1 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 de-stain it in 100 milliliters of 10% ethanol for 30 minutes. Then, rinse the gel in ultrapure water for 5 minutes. The gel can be visualized on a benchtop trans-illuminator, or imaged on a gel documentation machine at the 365-nanometer channel or the 302-nanometer channel.

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Silver Staining of Sulfated Glycosaminoglycans: A Staining Method for Visualization of Sulfated Glycosaminoglycans in Polyacrylamide Gels

Once the run is complete, disassemble the cassette, and carefully extract the gel into a large, clean container filled with deionized, filtered water. Then, discard the water and stain the gel in Alcian blue staining solution for five minutes. Discard the Alcian blue stain, and quickly wash two to three times with deionized, filtered water until most of the Alcian blue staining solution has been removed.
Stain the gel in silver nitrate staining solution in a fresh, clean container for 30 minutes. Then wash two to three times for 30 minutes each in deionized filtered water, to fully remove the silver staining solution. Discard the water, and add developing solution.
Carefully observe the gel for the appearance of bands. Depending on the quality of the stain and the mass of the sample loaded, development can take anywhere from a few seconds to several minutes. As soon as the desired bands are visible, immediately discard the developing solution, and wash the gel briefly with stop solution.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

Prepare two separating gels as described in the text manuscript. Pour the gel between the glass plates using a 1-milliliter pipette, ensuring that the upper 2 centimeters are free of the mixture. Add 70% ethanol on top of the separating gel, creating an even interface between the two layers.
Once the separating gels have polymerized, prepare the stacking gels using instructions in the text manuscript. Then, remove the ethanol from the separating gels, and add the stacking gel solution. Carefully insert a comb with the desired number of pockets, without introducing air bubbles, and allow the gel to polymerize for 20 to 30 minutes.
Load 4 microliters of each sample, as well as the protein ladder, into separate wells. Then, run the gel in Tris-Glycine running buffer at 144 volts for 45 minutes at room temperature. Use pre-warmed Fairbanks solution A to stain the gels on a rocker for 30 minutes, and pre-warmed Fairbanks solution D to decolor the gels on a rocker until the desired background is achieved.

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

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