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.
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).
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.
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.
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.
Monday – Friday
9 AM – 5 PM EST
DISCLAIMER: ConductScience and affiliate products are NOT designed for human consumption, testing, or clinical utilization. They are designed for pre-clinical utilization only. Customers purchasing apparatus for the purposes of scientific research or veterinary care affirm adherence to applicable regulatory bodies for the country in which their research or care is conducted.
