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Silver staining is a powerful technique for protein identification in gels as silver binds to chemical sidechains of the amino acids, including the carboxyl and sulfhydryl groups. It was introduced in 1972 and later adapted for protein separation from the polyacrylamide gel electrophoresis. 

The nucleation sites in proteins promote the reduction of silver ions by formaldehyde into microscopic grains of elemental silver, enabling their detection. The treatment does not modify the tertiary structure of the proteins. 

It has widespread applications owing to its high sensitivity, which typically is two orders of magnitude greater than the Coomassie and Ponceau staining methods and could even detect as little as 2 ng protein.

There are two main protocols for silver staining: alkaline and acidic, depending on the silver impregnation. The alkaline method uses a diamine complex of silver nitrate with an alkaline environment (ammonia and sodium hydroxide). 

And, then the patterns are developed in dilute acidic solutions of formaldehyde. The acidic method uses silver nitrate solution in water for gel impregnation and pattern development in formaldehyde solutions under alkaline conditions. 

The silver staining allows increased peptide coverage with higher sensitivity, reduced background, and least mass spectrometry interference.

Properties

  • Melting point: 93 K ​(961.78 °C, ​1763.2 °F)
  • Boiling point: 2435 K ​(2162 °C, ​3924 °F)
  • Density: 10.49 g/cm3
  • Heat of fusion: 28 kJ/mol
  • Heat of vaporization: 254 kJ/mol
  • Molar heat capacity: 25.350 J/(mol·K)

Principle

The principle of the technique is simple and based on silver reduction at the initiation site closer to the protein molecules. Silver staining starts with the fixation step in which proteins are immobilized, and interfering compounds are removed. The gel is then treated with compounds that either makes the proteins reactive to silver or accelerate the silver reduction. After that, silver impregnation is performed using either plain silver nitrate or ammoniacal silver. Finally, the gel is rinsed, and the silver metal image is obtained. Depending upon the amount of silver attached to the protein bands, different shades in the gel is produced (Kumar., 2018).

Protocol (Fernandez-Patron C. et al. 1992)

SDS-PAGE

  1. Add 20 μg of protein in 10 μL of sample buffer and leave it for 60 minutes at room temperature before separation.
  2. Fill 8 mL each of 3% acrylamide solution (Mix 2.0 mL 0.8 M Tris-HCl, pH 8.6, 0.75 mL 38.9% (w/v) acrylamide and 1.1% (w/v) bisacrylamide in 7.25 mL H2O, and 8 mg ammonium persulfate) and 20% acrylamide solution (Mix 2.0 mL 0.8 M Tris-HCl, pH 8.6, 5.0 mL 38.9% acrylamide and 1.1% bisacrylamide in 3 mL H2O, and 8 mg ammonium persulfate) in a gradient mixer and pump it with a flow rate of 5 mL/min into a glass cuvette.
  3. Load the protein samples in the gel and use phenol red as a tracking dye.
  4. Run the SDS-PAGE gel in a Protean II chamber (Bio-Rad) at 4 °C and 15 mA current for electrophoresis.
  5. Calculate the protein concentration using bovine serum albumin as per the Bradford method.

Silver staining

  1. Fix the gel in fixation solution (40% ethanol, 10% acetic acid, 50% water) for 30 minutes.
  2. Treat the gel with protein treatment solution (20% ethanol, 5% acetic acid, 75% water, 4 mg dithiothreitol) for 30 minutes.
  3. Rinse the gel with 0.5% dichromate for 5 minutes.
  4. Wash the gel with water for 5 minutes.
  5. Equilibrate the gel with 0.1% silver nitrate for 30 minutes.
  6. Briefly wash the gel with water for 1 minute.
  7. Incubate the gel in complex formation solution 0.02% paraformaldehyde, 3% sodium carbonate (Na2CO3), pH 12.
  8. Add 1% acetic acid to stop the complex formation.
  9. Fix the gels onto glass or polyester sheets for indefinite storage.

Long silver nitrate staining (Chevallet., Luche., & Rabilloud., 2006)

  1. After electrophoresis, fix the gels in 30% (v/v) ethanol and 10% (v/v) acetic acid for 60 minutes, then renew the fixation bath and leave overnight.
  2. Sensitize for 45 minutes in tetrathionate sensitizing solution.
  3. Rinse with 20% ethanol for twice, 10 minutes for each wash.
  4. Rinse four times with water, 10 minutes for each wash.
  5. Impregnate the gel with 12 mM silver nitrate.
  6. Arrange the gels soaking in silver nitrate, a box half-filled with water, basic developer, and a box containing the stop solution (40 g of Tris and 20 ml of acetic acid per liter). Rinse with deionized water, and pull the gel out of the silver solution.
  7. Dip it for 10 seconds in the water bath, then transfer it to the basic developer solution. Redissolve the precipitates by shaking off the developer-gel containing box.
  8. When the adequate degree of staining is achieved, transfer the gel to the Tris-stop solution for 30 minutes.
  9. Wash the gel with water and store.

Aldehyde-free silver-ammonia staining

  1. Fix the gels in 30% ethanol (v/v), 10% acetic acid (v/v) and 0.05% (w/v) naphthalene disulfonic acid. Repeat the fixation for three times, 30 minutes each.
  2. Rinse the gels in water six times, 10 minutes each.
  3. Impregnate for 30-60 minutes in silver-ammonia solution.
  4. Rinse with water three times, 5 minutes each.
  5. Develop image (5-10 min) in the acidic developer.
  6. Stop development in EA stop solution (0.5% (v/v) ethanolamine and 2% (v/v) acetic acid). Leave in this solution for 30-60 minutes.
  7. Rinse with water (several changes) before drying.

High-sensitivity silver-ammonia staining with formaldehyde fixation

  1. Rinse the gel with water for 5-10 minutes.
  2. Soak the gels in 20% (v/v) ethanol containing 10% (v/v) formalin for 60 minutes.
  3. Rinse twice with water, 15 min each rinse.
  4. Sensitize in 0.05% (wt/v) naphthalene disulfonate overnight.
  5. Rinse with water six times, 20 minutes each rinse.
  6. Impregnate for 30-60 minutes in silver-ammonia solution.
  7. Rinse with water three times, 5 minutes each rinse.
  8. Develop the image (5-10 minutes) in the acidic developer.
  9. Stop image development in EA stop solution (0.5% (v/v) ethanolamine and 2% (v/v) acetic acid). Leave for 30-60 minutes.
  10. Rinse with water (several changes) before drying.

Applications

1. Visualization of the regional variation of proteins (Merril., Goldman., Sedman., & Ebert., 1981)

The silver staining method is a rapid and easy-to-use technique for the detection of as little as 0.01 nanogram of protein per square millimeter. Along with the two-dimensional electrophoresis, it allows qualitative and quantitative characterization of protein distributions in body fluids and tissues. In the study, it was used to demonstrate the regional variations of cerebrospinal fluid (CSF) proteins. Marked differences in the distribution of several proteins, including albumin, immunoglobulin G, G,-globulin, al-antitrypsin haptoglobin, cx2Hs-glycoprotein, and transferrin, were observed. 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 subregions of CSF. The silver staining was found to be a valuable technique to study the regional variations of proteins in body fluids.

2. Detection of lipopolysaccharides in polyacrylamide gels (Fomsgaard., Freudenberg., & Galanos., 1990)

Silver staining technique is widely used for the detection of bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels. In the study, the method was modified to stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS and partly deacylated (alkali-treated) S-form LPSs. By omitting the fixation step, which was initially developed for detecting proteins, and by increasing the LPS oxidation time, the ability of silver stain to detect LPS fractions was increased. This modified procedure enables a fast, simple, and sensitive analysis of bacterial lipopolysaccharides in polyacrylamide gels.

3. Genomic and proteomic analysis

The silver stain was initially used for proteins, but later it was adapted for the staining of DNA and RNA. The staining does not interfere with the tertiary structure of the polymers and downstream analytical processes. 

Quantitative, as well as qualitative characterization of proteins, could be performed using the silver staining technique. 

The staining method is considered to have 100-fold more sensitivity than the Coomassie blue. This method is usually preferred to detect minor contaminants in purified proteins or lower concentrations of proteins.

Precautions

  • Do not touch the gel with bare hands as silver stain could detect keratin proteins in the skin.
  • Fix the proteins before staining to denature the proteins and prevent diffusion.
  • For simple proteins, fixation should be done with 80% methanol.
  • Duration of silver impregnation is critical to the color formation by gels.
  • Use freshly prepared buffers that contain aldehydes.
  • Perform the staining at room temperature with gentle agitation of the gels in a glass container.

Strengths and limitations

  • The silver staining technique is rapid, sensitive, and easy-to-use staining method for the identification of proteins separated on gels.
  • Silver stain provides 100-fold increased sensitivity as compared to other protein identification stains.
  • The silver staining 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.
  • The method can be efficiently used to stain proteins, DNA, and RNA.
  • The limitations of the silver stain include high and erratic background and the extreme protein to protein variability in staining.

References

  1. A. Fomsgaard., M. A. Freudenberg., & Galanos., C. (1990). Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol, 28(12), 2627–2631.
  2. , G. (2018). Principle and Method of Silver Staining of Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Methods Mol Biol, 1853, 231-236.
  3. Chevallet., S. Luche., & Rabilloud., T. (2006). Silver staining of proteins in polyacrylamide gels. Nat Protoc, 1(4), 1852-8.
  4. C. Merril., D. Goldman., A. S. Sedman. & Ebert., H. M. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science, 211(4489), 1437-8.