BMBMethod:silver

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Silver Staining of PAGE gels

• Fix the gel in fixing solution (50% methanol, 12% acetic acid, 0.5mL/L 37% formaldehyde) for 45min to overnight. (Gel can be kept in this solution for a couple of days without affecting the quality.) Change the solution 2 to 3 times during fixation.
• Wash the gel with 50% ethanol
  • 3 times, 20 min
  • OR
  • 4 times, 8 min each [GolbeckShort=4x8min].
  • If the gel is less than 1mm thick, the last wash is in 30% ethanol.
• Pretreat the gel in Na₂S₂O₃•H₂O (0.2g/L) for 1 min.
• Rinse with ddH2O for 3 times, 20 sec each.
• Incubate the gel in the solution of AgNO₃ (2g/L), 0.75ml/L 37% Formaldehyde for 20 min.
• Rinse with ddH₂O for 2 times, 20 sec each.
• Incubate the gel in developing solution (60g/L NaCO₃, 0.5ml/L 37% Formaldehyde, 4mg/L Na₂S₂O₃•H₂O) until desired intensity of protein spots.
• Wash with ddH₂O for 2 times, 2 min each.
• Stop the reaction in fixing solution for 10 min.
• Wash the gel with 50% methanol for at least 20 min. The gel can be kept in this solution at 4ºC.

Notes

• Most solutions should be made fresh
  • Pretreatment (Na₂S₂O₃•H₂O)
  • Silver stain (AgNO₃)
  • Developing solution
• All of the above steps are carried out on a shaker at RT.

Reagents
Good for 1 mini-gel (6x8cm)

• (~50mL (4x12.5)) Fixing: 50% MeOH, 12% Acetic Acid, 0.5mL/L 37% Formaldehyde
• (~50mL) 50% EtOH
• (10-15mL) 50% MeOH

MAKE FRESH

• (10-15 mL) Sodium Thiosulfate (0.2g/L)
• (10-15mL) Staining: 2g/L AgNO3, 0.75mL/L 37% Formaldehyde
• (10-15mL) Developing: 60g/L NaCO3, 0.5mL/L 37% Formaldehyde, 4mg/L Sodium Thiosulfate)