Molecular Biology. Practical Guide - Velikov V.A. 2013

Protein Electrophoresis
Coomassie R-250 Protein Staining

Processing polyacrylamide gels after Electrophoresis involves staining and destaining to remove unbound dye. The gel is immersed in trichloroacetic acid, thoroughly washed with Water, and placed in a staining solution of Coomassie Brilliant Blue R-250.

Dye molecules bind to Arginine and hydrophobic amino acid residues. The bound form is blue, so protein bands are visible on the gel with varying staining intensity that correlates with the protein concentration.

After staining the protein bands, the gel must be destained to remove unbound Coomassie R-250 dye. Gels are destained in solutions of ethanol (or methanol - toxic!) and acetic acid.

Materials and Equipment

Polyacrylamide gel with protein electrophoresis results. Solutions

- 10% TCA (Exercise 11.1).

- Coomassie R-250 solution (Exercise 11.1).

- Destaining solution I (Exercise 11.1).

- Destaining solution II (Exercise 11.1).

Procedure

1. Immediately after electrophoresis, place the gel in a destaining container, add 10% TCA, and leave for 30–60 min. TCA can be replaced with 5 mM HCl.

2. Add water, rinse thoroughly, and discard the water.

3. Transfer the gel to the Coomassie staining solution ^a for 1–1.5 h.

4. Discard the Coomassie solution and add destaining solution I ^б for 1–1.5 h.

5. Remove destaining solution I.

6. Place the gel in destaining solution II and leave it until the Background disappears from the gel. During the gel development process, it is recommended to replace destaining solution II 2–3 times.

Notes

^a Protein Staining with silver ions provides an order of magnitude higher sensitivity. After treating the gel with acetic acid and ethanol in solution I, thorough washing with ethanol and water, the gel is transferred for 30 minutes into 5 volumes of a 0.1% AgNO₃ solution. Then, after rinsing with water, it is placed in the same volume of 0.28 M sodium carbonate supplemented with up to 0.02% formaldehyde. Stained protein bands appear within a few minutes. Wait until optimal contrast is achieved and stop the reaction by rinsing in 10% acetic acid.

^б Alternatively: the gel is covered with cold water (200–300 ml) and boiled for ~5'