Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Summary

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Abstract

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

Protocol

Part 1: Preparation of the CBB staining solution

1. 60-80 mg of CBB G-250 are dissolved in 1 liter of bidistilled water by stirring for 2-4 hours. Finally, 3 ml of concentrated HCl is added to the dark blue solution with stirring for another minute and stored in the dark for later use. The solution can be stored for weeks up to several months without losing its staining efficiency.
2. Concentrated HCl should be handled with the usual care und used under a fume hood. The final solution will be at about pH 2, so gloves should be used and any contact with the skin should be avoided.

Part 2: SDS-PAGE

1. Appropriat....

Discussion

• The washing steps are critical for the efficient staining of the proteins. A reduced washing time below 2 min or reduced volume of water (<50ml) can result in pale blue protein bands, most likely due to higher amounts of residual SDS in the gel.
• If the proteins are going to be analysed by mass spectrometry, the heating steps in the microwave oven should be skipped, the time for washing of the gel extended to about 10 min in each step and the staining time extended until the band intensity is strong enough. Heating the gel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coomassie Brilliant Blue G-250	AppliChem	A3480	any other CBB G-250 could be used as well
Concentrated HCl