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

Abstract

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 staining method of polyacrylamide gels for the visualization of total protein by using fluorogenic probes.

Protocol

1. Preparation of the Gel

1. 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.
2. Dilute the samples with a mixture of distilled water, lithium dodecyl sulfate (LDS) buffer, and a sample-reducing agent.
3. Load the first lane with double the amount of stock (10 µL), followed by the normal stock amount (5 µL) and a series of twofold dilutions of the stock thereafter (13 dilutions, from 2x to 8192x).
4. Run the gel at a constant voltage of 200 V for 30 min.

2. Fixation of the Gel

1. 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 °C.
2. 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.

3. Preparation of the AgNO₃ Solution and Silver Impregnation of the Gel

1. 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 µL 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.
2. 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.
3. Wash the gel with ultrapure water (about 100 mL) in a clean container for 2 x 60 s.

4. Fluorogenic Development of the Gel

1. 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.
2. 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 TPE-4TA was synthesized following the protocol recently reported by Xie et al. The TPE-4TA solution is very stable and can be kept in the dark for months.
3. To prepare 100 mL of the fluorogenic developing solution (10 µM), 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 µM).
4. 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 °C for staining and then leaving it at room temperature.

5. Destaining and Imaging

1. 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.
2. Rinse the gel in ultrapure water for 5 min.
3. Image the gel at the 365 nm channel or 302 nm channel by a gel documentation machine.

Materials

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