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)