A Rapid Ethanol-Based Coomassie Blue Statining Protocol for SDS-PAGE Gels

[domba]

Author: Roger Rowlett, Colgate University.

Rapid ethanol-based Coomassie Blue staining of SDS-polyacrylamide gels
This protocol was cooked up from a combination of information provided by Novex (electrophoresis products) and by some of my colleagues at NIH, where I was on sabbatical leave last year. The protocol should also work for large format gels (250 x 250 x 1.5 mm gels) as well, but I haven't tried it myself. It also works well with methanol-based stain and destain, but significant amounts of methanol vapor are released into the laboratory---the ethanol is safer and just as effective. -Roger Rowlett

Procedure for developing 100 x 100 x 1 mm PAGE mingels

1. Place minigel in a loosely covered glass or microwaveable plastic (e.g., tupperware) container, ideally on top of plastic mesh if available.

2. Cover gel with 250 mL of staining solution.*

3. Microwave loosely covered gel/stain on high for approximately 2 minutes or until the solution just begins to boil. (Gels of 10-12% acrylamide are quite robust and will not be damaged even if the solution is boiled for a few minutes.)

4. Place the loosely covered gel/stain container on a slow shaker or simply leave on the bench for 15-60 minutes. It is usually possible to discern bands after as little as 15 minutes.

5. Remove stain from the container (it can be reused many times) and rinse the gel and gel container with water to remove excess staining solution.

6. Cover gel with 200-250 mL of detaining solution (staining solution minus dye).

7. Microwave on high for approximately 2 minutes or until the solution just begins to boil.

8. Place the loosely covered gel/stain container on a slow shaker or simply leave on the bench for 1-24 hours. For rapid destaining, change the destaining solution after one hour and microwave. One to three changes are usually sufficient to visualize bands with a clear background (2-4 hours total destaining time). For overnight destaining, place one large or several small crumpled Kimwipe tissues in the detaining solution to bind up dye, and agitate on a slow shaker.

9. Destained gels are rinsed throroughly with and stored in distilled water. Rinsed gels can be immediately dried in a membrane air-dryer for longer term preservation.

* Low-toxicity Staining solution:

0.25 g Coomassie Blue R-250
100 mL ethanol
100 mL water

Stir until dye is completely dissolved, about one hour. Add 25 mL acetic acid and make to 250 mL with water. Store at room temperature in a dark bottle. The final solution is 0.1% Coomassie blue, 10% acetic acid, 40% ethanol.

The destaining solution is prepared similarly, but without dye. The original recipe is:

400 mL ethanol
100 mL acetic acid

make to 1000 mL with water. Store at room temperature.

Bonus tip: 1.0 mm SDS-PAGE minigels run in Tris-glycine buffers can be safely run at 250 V constant voltage (twice the recommended voltage) without any degradation in separation quality. This reduces running time from 90 minutes to about 40 minutes in our hands. Novex claims you can go as high as 300 V, but I haven't had the inclination to try that with my homemade gels.

Decrease toxicity of destain even more!
The destain procedure can be made even less toxic by replacing the destaining solution completely with MilliQ water and no organic solvents. Basically, a freshly stained gel is immersed in water then microwaved on high for 15 min for a 0.75 mm thick gel or 25 min for 1.5 mm thick gel.

The method based on from:

G. Hervieu, MRC Molecular Neuroscience Group, Department of Neurobiology, Babraham Institute, Cambridge, UK CB2 4AT.