Staining Protein Gels with Coomassie Blue

By National Diagnostics | September 16, 2011 | Comments Off on Staining Protein Gels with Coomassie Blue

The Coomassie dyes (R-250 and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. Coomassie R-250, the more commonly used of the two, can detect as little as 0.1 ug of protein. Though less sensitive, Coomassie G-250 can be used in place of the R-250 form to create a rapid and convenient staining procedure. This capability of G-250 is due to its particular properties. Coomassie G-250 manifests a leuco form below pH 2. Solutions of the dye, dark blue black at pH 7, turn a clear tan upon acidification. The leuco form recovers its blue color upon binding to protein, apparently due to the more neutral pH of the environment around the protein molecule. Under proper conditions, a gel placed in an acidified solution of Coomassie G-250 will manifest blue protein bands on a light amber background. The bands develop rapidly and there is no need to destain, for the background color is so light as to be essentially clear. This stain is less sensitive than Coomassie R-250 protocols, detecting 0.5 µg of most proteins. The loss in sensitivity is offset by the speed and convenience of the protocol, which saves up to 11 hours versus the most sensitive R-250 procedures.

Coomassie blue R-250

Coomassie Blue G-250The two Coomassie blue dyes, R-250 and G-250, differ by only two methyl groups. In function, the more popular Coomassie R-250 is more sensitive while G-250 is more convenient.

Staining Gels with Coomassie Blue R-250 or Coomassie Blue G-250

STANDARD PROTOCOL - COOMASSIE BLUE R-250

  1. Gel may be prefixed in 50% MeOH, 10% HoAC, 40% H2O for 30 minutes to overnight.
  2. Stain gel in the above solution, with 0.25% Coomassie Blue R-250, for 2 - 4 hours, until the gel is a uniform blue color. Staining is complete when the gel is no longer visible in the dye solution. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution.
  3. Destain for 4 - 24 hours in 5% MeOH, 7.5% HOAC, 87.5% H2O. Bands will begin to appear in 1 - 2 hours. Destain until background is clear. Note: This method will detect as little as 0.1µg/band.
  4. Store gels in 7% HOAC.
    Troubleshooting

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RAPID PROTOCOL - COOMASSIE BLUE R-250

  1. Fix gel in 25% IPA, 10% HOAC in water for 30 - 60 minutes.
  2. Stain gel in 10% acetic acid in water, containing 60 mg/L of Coomassie Blue R-250. Bands will appear in 30 minutes. Allow staining to proceed until desired band intensity is reached. In this protocol, background staining is low due to the very low dye concentration used.
  3. Destain gel in 10% acetic acid for 2 hours or more. Store gels in 7% HOAC.
    Troubleshooting

RAPID PROTOCOL - COOMASSIE BLUE G-250

  1. To make the Coomassie Blue G-250 staining reagent, dissolve 0.2g dye in 100 ml H2O (this will require warming to approximately 50°C). Cool and add 100 ml 2N H2S04. Incubate at room temperature 3 hours to overnight, then filter. To filtered solution, CAREFULLY add 22.2 ml 10N KOH, then add 28.7g TCA. Allow to stand > 3 hours, then filter again if necessary to obtain an amber-brown solution without blue precipitate.
  2. To stain, immerse gel in above solution. Bands will begin to appear within 15 minutes. Intensity and sensitivity will continue to improve for several hours.
  3. Staining solution is stable for 2 - 3 weeks @ 25°C.
    Troubleshooting

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