Preparation of Coomassie Stain

Coomassie Stain - 1 L

0.1% Coomassie R250, 10% acetic acid, 40% methanol

Reagents needed:

1 g	Coomassie R250
100 mL	glacial acetic acid
400 mL	methanol
500 mL	ddH2O

Directions:

1) Add 100 mL of glacial acetic acid to 500 mL of ddH2O.

2) Add 400 mL of methanol and mix.

3) Add 1 g of Coomassie R250 dye and mix.

4) Filter to remove particulates (a coffee filter works great for this and is cheap)

5) Store at room temperature in a sealable container.

De-Stain for Coomassie - 1 L

20% methanol, 10% acetic acid

Reagents needed:

200 mL	methanol
100 mL	glacial acetic acid
700 mL	ddH2O

Directions:

1) Add 100 mL of glacial acetic acid to 700 mL of ddH2O.

2) Add 200 mL of methanol and mix.

3) Store at room temperature in a sealable container.

Coomassie Staining and Destaining

Reagents needed:

• Coomassie stain solution

• Destain solution

• Kimwipes

• Microwave oven (optional)

Directions:

1. Remove SDS-PAGE gel from the glass and rinse once in ddH2O in a suitable container with a lid. Try not to use a container much larger or much smaller than the gel.

2. Add enough Coomassie stain to cover the gel by 1/2 in. (~1.5 cm).

3. Microwave on high power for 40 seconds to 1 minute (until the Coomassie stain boils).

4. Incubate the gel in the Coomassie stain for 5 to 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.

5. Pour off the Coomassie stain. The Coomassie stain can be recycled a couple of times by filtering it.

6. Rinse twice in ddH2O or used destain solution to remove the Coomassie stain from the container.

7. Add fresh destain solution to cover the gel by 3/4 in. (~2 cm).

8. Tie Kimwipes in a simple knot and place 4 of them in the destain solution around the gel. Try not to avoid laying the Kimwipes on the gel as this will cause uneven destaining.

9. Microwave on high power for 40 seconds to 1 minute (until the destain boils).

10. Incubate the gel in the destain solution for 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.

11. Discard the stained Kimwipes and replace them with fresh knotted Kimwipes.

12. Incubate a second time for 10 minutes overnight on a rocking table. Stop whenever the level of destaining is sufficient. Microwave again to speed up the process.

13. The used destain solution can be recycled a couple of times by storing it in a sealed container with sponges or Kimwipes to remove all traces of Coomassie stain.