fluorescent silver staining: a technique to visualize total protein in polyacrylamide gels

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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1. Preparation of the Gel
<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&#160;TPE-4TA&#160;will be activated by the acid and lead to strong background fluorescence in the gel.</li>
</ol>
3. Preparation of the AgNO3&#160;Solution and Silver Impregnation of the Gel
<ol>
	<li>To make the AgNO3&#160;solution (0.0001%) for the impregnation, first, dissolve 0.01 g of AgNO3&#160;in 10 mL of ultrapure water to prepare a 0.1% AgNO3&#160;stock solution. Next, add 100 &#181;L of the 0.1% AgNO3&#160;stock solution into 100 mL of ultrapure water to make the working solution. 
	NOTE: The AgNO3&#160;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 aluminium 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&#160;TPE-4TA&#160;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 non-emissive solutions indicate full dissolution. 
	NOTE: The dye&#160;TPE-4TA&#160;was synthesized following the protocol recently reported by Xie&#160;et al.&#160;The&#160;TPE-4TA&#160;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&#160;TPE-4TA&#160;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.&#160; 
	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>

<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&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>

Source: Wong, A. Y. H. et al.<a href="https://www.jove.com/v/58669"> Fluorescent Silver Staining of Proteins in Polyacrylamide Gels.</a> J. Vis. Exp. (2019)
In this video, we demonstrate a silver staining method of polyacrylamide gels for the visualization of total protein by using fluorogenic probes.

Source: Wong, A. Y. H. et al. Fluorescent Silver Staining of Proteins in Polyacrylamide Gels. J. Vis. Exp. (2019)
In this video, we demonstrate a silver ...

Fluorescent silver staining of proteins in polyacrylamide gels employs fluorogenic probes to detect and quantify the proteins.
Begin with a polyacrylamide gel with proteins, separated into bands through electrophoresis. Soak in a fixative mixture of ethanol and acetic acid, which prevents the bands from diffusing by denaturing the proteins.
Wash the gel to remove residual acetic acid and ethanol. Immerse the gel in silver nitrate stain. Silver ions bind strongly to negatively charged groups in proteins.
Incubate in the dark. Rinse the gel in ultrapure water to remove unbound silver ion complexes.
Immerse the gel in a developing solution containing a multi-component fluorogenic probe - TPE-4TA. This anionic probe targets silver ions bound to the protein, forming insoluble aggregates that activate the fluorogenic properties of the probe, thus imparting fluorescence.
As the fluorescence activation is aggregation dependent, the unbound probes do not emit any signal, enabling total protein staining with reduced background emission.
Incubate the gel in the dark with gentle agitation, ensuring complete fluorogenic development. Transfer the gel in a destain buffer to remove unbound probes. Finally, image the gel to obtain the band signal intensity. Plot the fluorescence signal intensity against standard protein curve to calculate the total amount of proteins in the sample.

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

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