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.
