12.  Silver  Staining 

A. Materials :

      a.   Fixative I:

Methanol                                                          -     80 ml

Glacial acetic acid                                           -     20 ml

Mix and make up to 200 ml

      b.   Fixative II:

Ethanol                                                             -     40 ml

Glacial acetic acid                                           -     20 ml

Mix and make up to 400 ml

      c.   Oxidiser (3.4 mM potassium dichromate in 3.2 mM nitric acid.):

Dissolve 200 mg potassium dichromate in 150 ml double distilled water. Add 43 ml conc. nitric acid and make the volume to 200 ml with double distilled water.

      d.   Silver reagent (12 mM Silver nitrate):

            Dissolve 410 mg Silver nitrate in 100 ml double distilled water.

      e.   Developer (280 mM sodium carbonate in 0.05% formalin):

Dissolve 14.85 g sodium carbonate and 250 ml of formalin (37% w/v formaldehyde) in 500 ml double distilled water. 

B. Method :

1. Fix gel immediately after electrophoresis in 200 ml Fixative I for 30 min.

2.Transfer the gel to 200 ml of Fixative - II for 30 min with 2 changes. 

3.Immerse the gel in 200 ml of oxidizer for 10 min.

4.Put the gel in 400 ml of double distilled water. Change water thrice at 10 min intervals.

 5.Transfer the gel in 200 ml of Silver reagent for 30 min.

 6.   Immerse the gel in 400 ml of double distilled water for 5 min. Repeat this step.

7. Put the gel in 200 ml developer for 1 min.

8.Immerse the gel in fresh 200 ml developer for 5 min.

 9.Transfer the gel again in fresh 200 ml developer for about 5 min.

10. Stop  development  in  5%  acetic  acid  for  15  min  and  transfer  to  water  and then to preserving solution. 

Reference :

The BIORAD Silver stain-Bulletin 1089 (1982) 

Note :

 1. Use double distilled water for reagents preparation.

  2.Wear gloves throughout the procedure.

  3.If  the  bands  are  diffused,  miniaturization  of  the gel will help to get sharp bands. For miniaturization incubate the stained (coomassie or silver stain) gel in 50% PEG solution (in distilled water) for 2 hrs and then dry the gel.