Silver Stain Protocol

 

Silver Stain is about 10 to 100 times more sensitive than Coomasie Blue Staining; at least 1ng protein using this protocol. Estimation of protein amouts is not advisable since the staining intensity depends of the developing time and vary from protein to protein. Caution: nucleic acids are stain.

Fresh Solutions

0.02%  Sodium Thiosulfate (Na2S2O3)
0.1% AgNO3 for minigel and 0.15% for large gel. Stored in 4ºC before use.
2% Na2CO3 + 0.04% formaldehide. Prepare fresh.

Note: Wear clean gloves when handling all materials. Take special care to touch only the same corner of the gel to prevent staining artifact. Clean glasses very carefuly when preparing the gel. Place gel in clean glass container before staining

Staining steps  (for minigels). Every step needs gently shaking.

Fixation: 200ml 50% methanol + 5% acetic acid for 20min or ON (1hour for large gels).
Rinse 1: 200ml 50% methanol for 10min  (20min for large gels).
Rinse 2: 200ml H2O for 10min (15min for large gels).
Sensitization: 200ml 0.02% Na2S2O3 1min.
Rinse:H2O 2 x 1min.
Stain: 200ml chilled 0.1% AgNO3 for minigel (0.15% for large gel) for 20min. Keep from light.
Rinse: H2O 2 x 1min.
Develop: 2% Na2CO3 + 0.04% formaldehide with intensive shaking. After 20-30 seconds (solution turns yellow), replace it with new develop solution. Develop to the degree you want. You can change develop solution again after 2-3minutes.
Stop: 5% acetic acid 2 x 3min.
Store: 1% acetic acid at 4ºC, or rinse gel with same solution and then with H2O. If you need material for Mass Spectrometry cut the bands and store in 1.5ml plastic tubes at 4ºC.