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.
 

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