Western blot | Coomassie Blue Staining

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Coomassie Blue Staining

Table-1: Recipe of Staining Solution

Ingredients	Quantity
Coomassie Blue R-250	2.5 gm
Methanol	500 ml
Acetic acid	100 ml
DW	400 ml

Note: Final volume 1L.

1. Add 100 ml of acetic acid to 500 ml of ddH2O.
2. Add 500 ml of methanol and mix.
3. Add 1g of Coomassie R250 dye and mix.
4.  Put a magnetic bar in a brown bottle, and add dye while spinning.
5. Finally, filter it.

• Store at room temperature in a sealable container.

Table-2: Recipe of Destaining Solution

Ingredients	Quantity
Methanol	500 ml
Acetic acid	100 ml
DW	400 ml

• Store at room temperature in a sealable container.

Protocol

1. After the gel has finished running, remove it from the tank and remove the gel from the glass plates.
2. Wash the gel 2-3 times, 5 minutes each in deionized water to remove SDS present in the gel. Each wash should be large volumes of water.
3. Remove all free water from the gel. Add an adequate amount of staining solution (Coomassie Blue R-250) to cover the gel. And put a lid on the container to prevent the Coomassie from evaporation.
4. Place the container on a rocking device for gently shaking the gel in staining solution for 1 hour. Protein bands will be visible within 3-5 minutes and reach a maximum intensity within 1 hour. Longer incubation may be performed.
5. Rinse the stained gel in a large volume of deionized water, 2-3 times for 5 minutes each. To de-stain, cover the gel in destaining solution and place on a rocking device for 30 minutes or until desired resolution is attained.
6. Rinse the gel in deionized water and image/store the gel.

Note: If background staining is noticed, it is indicative of residual SDS in the gel. Rinsing the gel extensively in deionized water will remove the background staining.