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  Section: Plant Lab Protocols
 
 
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Methodology for Amino Acids and Proteins

 
     
 
Silver staining of proteins
 
The fractionated polypeptides on gels are visualized, by staining generally either with coomassie brilliant blue R 250 or amido black 10B dye. The above dyes can detect a band containing a little as 0.1mg of polypeptide. In may occasions, the available protein for electrophoresis is so small or some proteins occur in minute amounts the detection becomes extremely difficult with these dyes. Under such circumstances a higher sensitive detection system is required. Silver staining is a very useful method in this regard with about 100-fold greater sensitivity over dye staining. It is comparable in sensitivity to autoradiography of labeled polypeptides. There are different methods described by different workers for silver staining. The method given below is very simple and rapid.
 
 


Principle
The amino acids particularly aromatic amino acids in the protein reduce silver nitrate and form complexes with metallic silver of yellowish-brown to brown color.
 
 
 Materials
Washing solution
Mix 1mL of formaldehyde (analytical grade, 37%), 40mL of methanol and 60mL of distilled water and use.
Sodium thiosulphate: dissolve 200mg in a liter of water
Silver nitrate solution (0.1%)
Developer: 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.
Stopper: 5% citric acid or 5% acetic acid solution.
 
 
Procedure
1.
After electrophoresis, transfer the gel to a clean plastic container and wash the gel in the washing solution with slow shaking for 10min.
2.
Discard the wash solution and rinse the gel with plenty of water for 2min.
3.
Soak the gel in sodium thiosulphate solution for 1-2min.
4.
Wash the gel with water twice, each time 1-2min. drain the wash water.
5.
Soak the gel in silver nitrate solution for 10min with gentle shaking.
6.
Wash the gel in water as in step 4.
7.
Pour developer to the plastic container and shake the gel slowly, gently. The proteins reduce silver nitrate to silver and the yellow to dark brown color bands appear.
8.
When sufficient intensity of band developed stop the reaction by adding either citric acid or acetic acid solution.
9.
Record the protein banding pattern by photography.
 
 


Notes
1.
Cleanliness is very much important in this experiment since the reaction is very sensitive. Use filtered reagents. All glassware should be thoroughly cleaned.
2.
Wear gloves while working, particularly when handling the gel; otherwise fingerprints will be a problem.
3.
The gel becomes very fragile during the treatment, exercise adequate care.
4.
The method is very rapid and easy. The proteins can be fixed in the gel, if necessary, by immersing it in 10% glutaraldehyde for 30-60min and washed in water prior to washing the gel in washing solution mentioned above.
5.
Each washing step should be done effectively.
6.
Continuous gentle shaking of the gel in every step improves uniform staining.
7.
Destained and/or unstained gels can be used for detection of proteins. Destained gels are more rigid and can be handled easily than the unstained gels.
8.
The color formation is inorganic in reaction and hence proceeds very rapidly. Stop the reaction once the required intensity of bands is obtained by pouring stopper solution and with washing.
 
 


References
1. Oakley, B, Kirsch, D and Morris, N R (1980) Anal Biochem 105, 361.
2. Merril, C R, Danan, M and Goldman, D (1981) Anal Biochem 110, 201.
3. Poehling, H M and Neuhoff, V (1981) Electrophoresis 2, 141.

 
     
 
 
     




     
 
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