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Protocols

Coomassie blue gel staining protocol

  1. Incubate the gel with 0.5% Coomassie Blue G-250 prepared in 50% methanol and 10% acetic acid for 5 minutes.
  2. Discard stain and rinse briefly with MilliQ water to remove most of the residual stain in the glassware.
  3. Destain with 40% HPLC-grade methanol and 10% acetic acid, replacing the solution every 10-20 min until faint bands are observed. Kimwipes rolled up into balls can be added to speed up the destaining.
  4. Continue destaining with MilliQ water until bands are very clean. Usually we destain overnight in MilliQ water with several Kimwipes present. Bands/spots can now be excised and submitted for analysis.

SYPRO Ruby staining protocol

  1. Incubate the gel with 50% methanol and 7% acetic acid for 30-60 minutes.
  2. Color the gel in non-diluted SYPRO Ruby for 3 hours (or overnight).
  3. Destain the gel in 50% methanol for 1 minute.
  4. Continue destaining the gel in 10% methanol and 7% acetic acid for 30-60 minutes.
  5. Rince in water for 5 minutes 2 times.

Silver staining protocol (compatible with in gel tryptic digestion)

O'Connell-Stults' protocol

(For the original protocol, see Electrophoresis 1997;18(3-4):349-59. For general advice on silver staining, see Nat Protoc. 2006;1(4):1852-1858.)

Fixation
Fix the gel in 30% EtOH and 10% acetic acid for 30 minutes 3 times. Rinse in 20% EtOH for 10 minutes, and then rinse in water for another 10 minutes.
Sensitization
Sensitize the gel in sodium thiosulfate (2.0g/l). Rinse in water for 20 seconds 2 times.
Siver impregnation
Impregnate with silver nitrate (2.0g/l) for 30 minutes. Rince in water for 5-10 seconds.
Development
Develop in 37% formaldehyde (0.7ml/l), potassium carbonate (anhyd., 30g/l) and sodium tiosulfate(10mg/l) 3 minutes two times (or to desired intensity).
Stopping step
Stop the development in Tris base (50g/l) and 2.5% acetic acid for 1 minute.
Storage
Store in water.

Shevchenko-Mann's method

(For the original protocol, see Anal Chem. 1996;68(5):850-8. For general advice on silver staining, see Nat Protoc. 2006;1(4):1852-1858.)

Fixation
Fix the gel in 50% MeOH, 12% acetic acid (240ml/2l) and 0.05% formaldehyde (2.7 ml/2l) for two hours or overnight. Rinse in 35% EtOH (700ml/2l) for 20 minutes 3 times.
Sensitization
Sensitize the gel in 0.02% sodium thiosulfate (2ml at 20%/2l) for 2 minutes. Rinse in water for 5 minutes 3 times.
Silver impregnation
Impregnate with 0.2% AgNO3 (4g/2l) and 0.076% formaldehyde (4.1ml/2l) for 20 minutes. Rinse in water for 1 minute 2 times.
Development
Develop in 6% sodium carbonate (120g/2l), 0.05% formaldehyde (2.7ml/2l) and 0.0004% sodium thiosulfate (40μl/2l) for 2-15 minutes.
Stopping step
Stop the development in 50% MeOH and 12% acetic acid (20ml/2l) for 5 minutes.
Storage
Store in 1% acetic acid (20ml/2l) at 4°C.