Coomassie Blue Staining

The coomassie staining protocol described below is recommended for staining Invitrogen™ Novex™ Gels. You may use any Coomassie™ staining protocol of choice.

Materials Needed

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

SimplyBlue™ SafeStain Protocol

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.

1. 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.
   
2. 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.
   
3. 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.
   
4. 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.

Colloidal Blue Staining Kit Protocol

1. Prepare staining solution for a single gel as described in the table below. For two gels, double the volume of reagents used for staining. Be sure to shake Stainer B prior to making the solution.