Introduction

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

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Materials Needed

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)
  • Methanol

 

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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 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.
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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.

  2. Solutions
       
    Solutions
                    Tris-Glycine, Tricine
    Deionized Water
    55 ml
    Methanol
    20 ml
    Stainer B
    5 ml
    Stainer A
    20 ml


  3. 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.

  4. 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.
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Coomassie® R-250 Staining Protocol

  1. Prepare the staining solution containing 0.1% Coomassie® R-250 in 40% ethanol, 10% acetic acid.

  2. 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.

  3. 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.

  4. Remove the staining container from the microwave oven and gently shake the gel for 15 minutes at room temperature on an orbital shaker.

  5. Decant the stain and rinse the gel once with deionized water.

  6. Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.

  7. Place one or two stained gels in a staining container containing the 100 ml destain solution.

  8. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute.

  9. Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved.
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