The coomassie staining protocol described below is recommended for staining Invitrogen™ Novex™ Gels. You may use any Coomassie™ staining protocol of choice.
Materials supplied by user:
You will need following items for Coomassie Blue Staining.
- Invitrogen™ SimplyBlue™ SafeStain
- Colloidal Blue Staining Kit
- Staining container
- Deionized water
- Orbital Shaker
- 0.1% Coomassie® R-250 in 40% ethanol and 10% acetic acid (if using Invitrogen™ Coomassie™ R-250 Staining)
- Destaining Solution consisting of 10% ethanol and 7.5% acetic acid (if using Invitrogen™ Coomassie™ R-250 Staining)
- Methanol
For general use with 1.0 mm and 1.5 mm Tris-Glycine gels, 1.0 mm Tricine, Zymogram, and IEF mini-gels (8 x 8 cm).
After electrophoresis follow the instructions below. Be sure the mini-gel moves freely in water or stain to facilitate diffusion during all steps.
- Rinse the mini-gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts, which interfere with binding of the dye to the protein. Discard each rinse.
- Stain the mini-gel with enough Invitrogen™ SimplyBlue™ SafeStain (20-100 ml) to cover the gel. Stain for 1 hour at room temperature with gentle shaking. Bands will begin to develop within minutes. After incubation, discard the stain. Stain cannot be re-used. Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity will decrease. If you need to leave the gel overnight in the stain, add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain. This procedure will not affect sensitivity.
- Wash the mini-gel with 100 ml of water for 1-3 hours. The gel can be left in the water for several days without loss of sensitivity. There is a small amount of dye in the water that is in equilibrium with the dye bound to the protein, so proteins will remain blue.
- To obtain the clearest background for photography, perform a second 1 hour wash with 100 ml water. Note: Sensitivity will now decrease if the gel is allowed to stay in the water more than 1 day. Reduction of free dye in the water favors dissociation of the dye from the protein. If you need to store the gel in water for a few days, add 20 ml of 20% NaCl.