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Silver Staining in SDS PAGE

In this post silver staining in SDS PAGE we briefly summarises about: principle, reagents requirements, silver staining protocol, result, application and limitations of silver staining protocol.

Silver Staining in SDS PAGE

Silver staining protocol is a highly sensitive method for detecting proteins and nucleic acids in polyacrylamide slab gels. The most sensitive colorimetric approach for detecting total protein is silver staining protocol. The method entails depositing metallic silver onto a gel’s surface at the positions of protein bands.

Silver ions (from the staining reagent’s silver nitrate) interact and bind with specific protein functional groups. Carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines have the strongest connections (Lys).

Controlling the selectivity and effectiveness of silver ion binding to proteins, as well as the successful conversion (development) of bound silver to metallic silver, requires a variety of sensitizer and enhancer reagents. Silver ions are converted to metallic silver throughout the development process, resulting in a brown-black hue.


The approach works on the simple premise of silver reduction at the start point, which is closer to the protein molecules. The fixation stage, in which proteins are immobilised and interfering chemicals are eliminated, is the first step in silver staining protocol. After that, the gel is treated with chemicals that either make the proteins reactive to silver or speed up the silver reduction process.

After that, either ordinary silver nitrate or ammoniacal silver is used for silver impregnation. Finally, the gel is washed to reveal the silver metal picture. Different colours in the gel are created depending on the amount of silver linked to the protein bands.


It has a boiling point of ​2162 °C (​3924 °F)

It has a melting point of 961.78 °C (​1763.2 °F)

The heat of vaporization: 254 kJ/mol

It has a density of 10.49 g/cm3

Molar heat capacity: 25.350 J/(mol·K)


Washing solution: Mix 1mL of formaldehyde (analytical grade, 37%), 40mL of methanol and 60mL of distilled water and use.

Sodium thiosulphate Solution: dissolve 200mg of Sodium thiosulphate in a liter of double distilled water

Developer Solution:  dissolve sodium carbonate 3g (w/v) in about 80mL water. Add 1mL of the above sodium thiosulpahte solution and 1mL of formaldehyde and finally make up the volume to 100mL with water.

Silver nitrate: This is usually used as a stock solution (typically 20% w/v), which must be stored in a dark place or can be purchased and used

Stop solution: 4% (w/v) Tris and 2% (v/v) acetic acid. CAUTION: must be prepared the day of use.

Silver Staining Protocol


1. Before separation, dissolve 20 µg of protein in 10 µl of sample buffer at room temperature for 60 minutes.

2. Fill 8 mL each of 3% acrylamide and 20% acrylamide solution in a gradient mixer and pump it with a flow rate of 5 mL/min into a glass cuvette.

3. Fill the gel with protein samples and apply phenol red as a tracking dye. For electrophoresis, place the SDS-PAGE gel at 4 °C and 15 mA current.

4. Calculate the protein concentration using bovine serum albumin as per the Bradford method.

Silver staining

1. For 30 minutes, soak the gel in a fixation solution (40 percent ethanol, 10% acetic acid, 50 percent water).

2. For 30 minutes, soak the gel in a protein treatment solution (20 percent ethanol, 5% acetic acid, 75% water, 4 mg dithiothreitol).

3. Rinse the gel for 5 minutes with 0.5 percent dichromate. For 5 minutes, soak the gel in water. For 30 minutes, equilibrate the gel with 0.1 percent silver nitrate.

4. For 1 minute, rinse the gel with water. Incubate the gel in a 0.02 percent paraformaldehyde, 3% sodium carbonate (Na2CO3), pH 12 complex formation solution.

5. To block the development of the compound, add 1% acetic acid. For long-term preservation, adhere the gels to glass or polyester sheets.

Long Silver staining

1. Fix the gels in 30 percent (v/v) ethanol and 10% (v/v) acetic acid for 60 minutes after electrophoresis, then repeat the fixation bath and leave overnight.

2. In a tetrathionate sensitising solution, sensitise for 45 minutes. Rinse twice with 20% ethanol, allowing 10 minutes between washes.

3. Rinse four times with water, each for ten minutes. 12 mM silver nitrate should be used to impregnate the gel.

4. Arrange the silver nitrate-soaked gels, a box half-filled with water, basic developer, and a box containing the stop solution in the correct order (40 g of Tris and 20 ml of acetic acid per liter). Pull the gel out of the silver solution after rinsing with deionized water.

5. Dip it in the water bath for 10 seconds before transferring it to the basic developer solution. Shake off the developer-gel-containing box to redissolve the precipitates.

6. Transfer the gel to the Tris-stop solution for 30 minutes once the desired level of staining has been obtained. Water should be used to clean the gel before storing it.

Silver Staining in SDS PAGE

Figure: Silver Staining Protocol in SDS PAGE


1. The silver staining protocol is a rapid and easy-to-use technique for the detection of as little as 0.01 nanogram of protein per square millimetre.

2. Along with the two-dimensional electrophoresis, silver staining protocol allows qualitative and quantitative characterization of protein distributions in body fluids and tissues.

3. Marked differences in the distribution of several proteins, including albumin, immunoglobulin G, G,-globulin, al-antitrypsin haptoglobin, cx2Hs-glycoprotein, and transferrin, were observed in silver staining protocol.

4. Some CSF proteins were found in lower concentrations in lateral ventricular CSF. However, other CSF proteins were not diminished in lateral ventricular CSF, suggesting that individual proteins vary in the sub-regions of CSF.

5. The silver staining protocol was found to be a valuable technique to study the regional variations of proteins in body fluids.

Advantages and Disadvantages

1. The silver staining protocol is rapid, sensitive, and easy-to-use staining method for the identification of proteins separated on gels.

2. Silver staining protocol provides 100-fold increased sensitivity as compared to other protein identification stains.

3. The silver staining protocol reaches the same performances in terms of identification as compared to the fluorescent probes, but its permanence and simplicity of spot cutting make it advantageous over fluorescent probes.

4. Silver staining protocol can be efficiently used to stain proteins, DNA, and RNA. The limitations of the silver staining protocol include high and erratic background and the extreme protein to protein variability in silver staining protocol.

Further Readings