Upload Now
In this article we will discuss about the principle, requirements and procedure for silver staining of proteins.
Polypeptides even upto 0.1 µg present on gel can be visualised by staining with amido black 10B dye or Coomassie brilliant blue R250. Silver staining is useful since some proteins occur in very small quantities and are difficult to be detected with these dyes. Silver staining is 100 times more sensitive than these dyes.
Principle:
Silver nitrate is reduced by amino acids, especially aromatic amino acids in protein, to form complexes with metallic silver which is yellowish-brown to brown in colour.
Requirements:
1. Silver nitrate solution 0.1%
2. Washing solution:
Analytical grade formaldehyd.
(37%): 1.0 ml
Methanol: 40.0 ml
Distilled water: 60.0 ml
3. Sodium thiosulphate 200.0 mg
Distilled water 1000 ml
4. Developer
Dissolve 3 g of (W/V) sodium carbonate in 80 ml distilled water. Add 1 ml of sodium thiosulphate solution and 1 ml of washing solution (formaldehyde) and makeup the volume to 100 ml with distilled water.
5. Stopper
Acetic acid-5% or Citric acid-5%.
6. Plastic container.
Procedure:
1. Wash the gel (electrophorised) with washing solution in a clean plastic tray for 10 minutes with mild shaking.
2. After discarding wash solution, wash the gel for 2 minutes with plenty of water.
3. Soak the gel for 2 minutes in thiosulphate solution.
4. Wash the gel twice for 2 minutes each, with water and drain.
5. Soak the gel in silver nitrate solution for 10 minutes with gentle shaking.
6. Wash the gel twice for 2 minutes each with water and drain off the water.
7. Pour developer to the plastic tray shake gently. Yellow to dark brown bands appear due to the reduction of silver nitrate to silver by proteins.
8. When there is sufficient intensity of band, stop the reaction by adding either acetic acid or citric acid.
9. Photograph the bands.