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

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.

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.