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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.
- 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)
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).