Colloidal Coomassie Staining
Sensitivity: 250 ng/mm2 or ca. 30 ng/band
Method A
Fixing solution
50% methanol
10% acetic acid
Gels are fixed for 2 hr to overnight. Ethanol may be used instead of methanol, but ethanol causes gel shrinkage. Use 250 ml of fix/stain/destain solution per 20 cm x 20 cm x 1.5 mm gel. Two gels may be handled together in a single tray.
| Staining solution |
|
|
Component |
Final Concentration |
Amount |
| |
|
|
| ammonium sulfate |
10% |
100 g |
| Coomassie G-250 |
0.1% |
1 g |
| MilliQ water |
-- |
800 mL |
| phosphoric Acid |
2% |
20 mL |
| methanol |
20% |
200 mL |
| Final Volume |
-- |
1020 ml |
Ammonium sulfate and Coomassie G-250 (also called Brilliant Blue G) are placed in a large flask with ~400 ml MilliQ water and stirred until the ammonium sulfate is dissolved. The phosphoric acid is then added, followed by small additions of methanol until all of the methanol is added. Then add the remaining water. There should be a fine colloidal suspension of Coomassie G-250.
Gels are stained for 2 hr to overnight on a platform shaker. Destain the gels using MilliQ water and Kimwipes twisted and placed in the tray to absorb the dye. Replace the Kimwipes 3 or more times during destaining as necessary
Method B
Gels can be placed directly into staining solution after electrophoresis with this method. Alternatively, gels can be fixed in 50% methanol-10% acetic acid (2 hr-overnight). Stain gels overnight; gels may be stained 2 hr if gels have been fixed first.
Component |
Final Concentration |
Amount |
| |
|
|
| ammonium sulfate |
17% |
85 g |
| Coomassie G-250 |
0.1% |
0.5 g |
| MilliQ water |
-- |
330 mL |
| phosphoric acid |
3% |
15 mL |
| methanol |
34% |
170 mL |
| Final Volume |
-- |
500 ml |
Solution A
Dissolve Coomassie Blue G250 in a minimum amount of methanol. Add phosphoric acid to the methanol/Coomassie blue solution.
Solution B
In a separate flask, dissolve ammonium sulfate in water. Slowly add methanol with stirring, a precipitate will form.
Slowly add the methanol/Coomassie blue solution A to the methanolic ammonium sulfate solution B. Pour this solution into the gel trays which are left on the shaker for 2 hours-overnight. Destain the gels using 10% acetic acid and Kimwipes twisted and placed in the tray to absorb the dye.
References
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248-54.
Top of page
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Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
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