Coomassie blue gel staining protocol
- Incubate the gel with 0.5% Coomassie Blue G-250 prepared in 50% methanol and 10% acetic acid for 5 minutes.
- Discard stain and rinse briefly with MilliQ water to remove most of the residual stain in the glassware.
- 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.
- 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
- Incubate the gel with 50% methanol and 7% acetic acid for 30-60 minutes.
- Color the gel in non-diluted SYPRO Ruby for 3 hours (or overnight).
- Destain the gel in 50% methanol for 1 minute.
- Continue destaining the gel in 10% methanol and 7% acetic acid for 30-60 minutes.
- Rince in water for 5 minutes 2 times.
Silver staining protocol (compatible with in gel tryptic digestion)
(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.)
- 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.
- 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.
- 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.
- Store in water.
(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.)
- 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.
- 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.
- 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.
- Store in 1% acetic acid (20ml/2l) at 4°C.