Ultimate CBB staining

Staining solution:

• 5% Aluminium sulfate 14-18 hydrate
• 10% Ethanol
• 0.02% CBB G-250
• 8% Phosphoric Acid

Note: for the preparation of the staining solution, the sequential addition of the components in the following order has to be maintained:

1. first dissolve aluminum sulfate in Milli-Q water
2. thereafter add ethanol, homogenize, and mix CBB G-250 to the solution
3. Let CBB dissolve properly for ~10min
4. as soon as the solution is completely dissolved, slowly add phosphoric acid (the incorporation of the acid to the alcoholic media lets the Coomassie molecules aggregate into their colloidal state)
5. finally fill up with Milli-Q water
6. Store in dark bottle

Note: the final staining solution has a dark green-bluish appearance and is full of particles swimming around.

• Do not filtrate this solution!
• If you changed the order of preparation, you would get a more violet-bluish solution that is less sensitive.

Staining procedure

• After running gels move them carefully into a staining dish filled with Milli-Q water.
• Wash the gels with Milli-Q water for 10 minutes. Note: do not wash too long as the protein might migrate out of the gel.
• Shake the Coomassie solution before use to disperse the colloidal particles evenly.
• Incubate the gels well covered with the Coomassie solution by agitation on a shaker for 2-12 hours. This staining generates marginal background so you can observe the staining progress in between. Occasionally turn the gels during the staining procedure so they are stained evenly from both sides (if you keep the gels unmoved, the dye just binds to one side of the gel). Note: after 10 minutes you can see first protein spots appearing, within 2 hours of incubation about 80% staining to its maximum level is completed. For nearly 100% staining, overnight incubation is recommended. In some cases, when the amount of protein to be stained is huge and the solution turns into a bright blue, a refresh of the staining solution is necessary.
• After the staining procedure remove the Coomassie solution and rinse the gels twice with Milli-Q water. Note: you can reuse the staining solution as long as particles are still remaining, otherwise discard it.
• Finally leave the gels in Milli-Q water: the gels will reach their original thickness (the alcohol-acid media makes the gels shrinking) and neutralization in water additionally enhances Coomassie’s color intensity.