The coomassie® staining protocol described below is recommended for staining Novex® Gels. You may use any Coomassie® staining protocol of choice.
Materials supplied by user:
You will need following items for Coomassie® Blue Staining.
- 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 Coomassie® R-250 Staining)
- Destaining Solution consisting of 10% ethanol and 7.5% acetic acid (if using Coomassie® R-250 Staining)
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 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.
- 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.
- Incubate the gel in this staining solution as follows at room temperature with gentle shaking:
- Tris-Glycine, Tricine, and Zymogram Gels for a minimum of 3 hours and a maximum of 12 hours .
- IEF Gels for 30 minutes.
- Decant staining solution and add a minimum of 200 ml of deionized water per gel to the staining container. Gently shake gel in water for at least 7 hours. Gel will have a clear background after 7 hours in water.
Note: Novex® Gels can be left in deionized water for up to 3 days without significant change in band intensity and background clarity.
For long-term storage (over 3 days), keep the gel in a 20% ammonium sulfate solution at 4°C.
- Prepare the staining solution containing 0.1% Coomassie® R-250 in 40% ethanol, 10% acetic acid.
- After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie® Blue R-250 staining solution. Caution: Use caution while performing the following steps using a microwave oven. Do not overheat the staining solutions.
- Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. To prevent hazardous, flammable vapors from forming, do not allow the solution to boil.
- Remove the staining container from the microwave oven and gently shake the gel for 15 minutes at room temperature on an orbital shaker.
- Decant the stain and rinse the gel once with deionized water.
- Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.
- Place one or two stained gels in a staining container containing the 100 ml destain solution.
- Loosely cover the staining container and heat in a microwave oven at full power for 1 minute.
- Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved.