Coomassie Blue Staining: Definition & Overview

Definition: Coomassie Blue Stains

Coomassie Brilliant Blue is a family of dyes that are routinely used in labs for protein staining protocols, performed after SDS-PAGE or polyacrylamide gel electrophoresis. The staining chemistry of these protein dyes is based on their binding with the protein and the matrix. 

The dye forms a complex with the basic amino acid residue of the proteins, including arginine, histidine, tyrosine, and lysine for their visualization. This binding imparts overall negative charges to the proteins and facilitates their separation from SDS-PAGE gels based on their molecular weight.

Coomassie Brilliant Blue has the potential to detect 30-100 ng proteins. However, its sensitivity depends on the type of stain (Coomassie Brilliant Blue G-250 and Coomassie Brilliant blue R-250) used in the staining method. 

After staining with the dye, you will observe blue bands with a blue background. The background noise should also be reduced to make the bands easier to visualize. This is done by using a destaining solution, which takes approximately 10 minutes to overnight to remove excess stain and produce bands with clear background.

Coomassie Brilliant Blue was first described by scientist Volker Neuhoff and got its name from an African city, Kumasi, which was previously known by the name of Coomassie. You must also know that the name “Coomassie” is a registered trademark of Imperial Chemical Industries.

The dye has a chemical formula C47H50N3NaO7S2 (Sodium salt) and a molar mass of 3 g/mol. It dissolves well in hot water and ethanol but slightly solubilizes in cold water. 

In this article, we will briefly cover a brief on types of Coomassie Brilliant Blue, their applications in lab assays, and industrial areas that extensively utilize the dye for a myriad of purposes.

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Types of Coomassie Brilliant Blue

Based on their properties, molecular characteristics, staining ability, and sensitivity, Coomassie Brilliant Blue is of two types: Coomassie Brilliant Blue G-250 and Coomassie Brilliant Blue R-250.

Coomassie Brilliant Blue G-250

Coomassie Brilliant Blue G-250 (or colloidal coomassie blue) contains two methyl groups, which are absent in the R-250 version of the dye. The “G” in the name denotes the greenish tint of the dye.

Figure: The structure of the Coomassie Brilliant Blue G-250.

Coomassie Brilliant Blue is available in three forms: cationic (red), neutral (green in color), and anionic (blue). In the presence of an acidic condition, the red coomassie dye converts to blue when it binds with the protein. However, when there is no binding, it remains brown.

During the complex formation of Coomassie Brilliant Blue with the protein, the reaction that occurs includes the following steps:

  • The dye transfers a free electron to the groups of the proteins that can be ionized.
  • The transfer of electrons to protein disrupts its native structure and exposes its hydrophobic pockets.
  • The non-polar components of the dye bind to the hydrophobic pocket of the protein through van der Waals forces.
  • The ionic interaction and complex formation through the reaction allow the visualization of the protein bands separated in protein gel.

The best commercial coomassie dyes are the ones that have high sensitivity, superior reproducibility, and low background. Moreover, they should allow flexible staining and destaining time for different lab assays.

Coomassie Brilliant Blue R-250

The Coomassie Brilliant Blue R-250 is another form of disulfonated triphenylmethane compound. The “R” in the name denotes the reddish tint of the dye. 

Coomassie Brilliant Blue R-250 was first used in 1963 by Fazekas de St. Groth and his colleagues to see proteins separated on a cellulose acetate sheet, after protein gel electrophoresis.

Figure: The structure of the Coomassie Brilliant Blue R-250.

The use of staining solution prepared with Coomassie Brilliant Blue R-250 is the fastest approach to stain polyacrylamide protein gels or IEF mini-gels. It’s of lower cost and provides better resolution than the G-250 form of the dye.

Applications of Coomassie Brilliant Blue

Coomassie Brilliant Blue staining is an efficient, simple, quick, and affordable protein gel staining technique. It can be routinely used in proteomics-related studies, such as mass spectrometry, SDS-PAGE, and IEF, to detect approximately 0.1–0.5 µg proteins. The protocol utilizes Coomassie Brilliant Blue R-250 in a methanol/acetic acid solution and stains proteins by gentle shaking at room temperature.

One other highly sensitive technique to visualize proteins is silver staining. However, it’s a cumbersome procedure because of the involvement of multiple steps and a number of solutions.

Below are two lab applications that are routinely performed and involve the use of Coomassie Brilliant Blue stain along with other reagents that are required to prepare the staining solutions.

Electrophoresis

  • Polyacrylamide (prepared by mixing acrylamide and bisacrylamide) gel electrophoresis is a technique to separate proteins based on their molecular weight for molecular biology experiments and protein analysis. The separated proteins can be detected by using either Coomassie Brilliant Blue, fluorescent, or silver staining methods.   
  • Coomassie blue is a fixation-free, fast, convenient, low-cost, easily operable, and widely used protein staining method in labs. Additionally, it’s also a more mass-spectrometry (MS) compatible method compared to other staining techniques. 

Assays

  • The determination of protein concentration is one of the essential experimental studies in many proteomics and protein studies. 
  • Coomassie Brilliant Blue is one of the widely used stains in protein estimation assays, such as the Bradford assay. It works on the principle of binding the dye with the protein molecules.

Industries that Use Coomassie Blue Staining

Being an efficient dye, the Coomassie Brilliant Blue is used in a range of labs and industries for several application purposes. Some of the areas that involve the use of the dye are mentioned below. 

Biochemistry

  • The G-250 form of the stain is used in the staining formulation with other reagents, such as ethanol (or methanol), phosphoric acid, and ammonium sulfate. It’s used to stain protein gels and in Bradford assay.
  • Moreover, in western blot analysis, the dye is used as a loading control staining method, where it’s employed as an anionic pre-antibody stain.

Medicine

  • Coomassie Brilliant Blue is a part of many experimental studies in labs, including treating spinal injuries. The treatment has been found effective in lab rats, however, their efficacy on humans is still unknown.
  • The dye also assists doctors in retinal surgery.

Forensic Science

  • The ability of Coomassie Brilliant Blue to form a complex with proteins enables its use in Bradford assay for fingerprint analysis.
  • It’s an efficient method to identify the biological sex of the fingerprint as the female samples show higher absorbance than male samples.
  • The technique is preferable, simpler, and easier than the ninhydrin chemical assay, which requires an assay preparation and involves the use of a cascade of enzymes in the process. 

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Coomassie Brilliant Blue is a widely used protein staining technique. After proteins are separated on the electrophoretic gels, they are stained using the staining solution containing Coomassie Brilliant Blue. The dye helps the visualization of proteins in the form of blue bands. 

However, during the staining procedure, the gel matrix is also stained. Therefore, to remove the background color, a destaining solution is applied to the gel for better visualization of blue protein bands on a clear background.

Apart from its uses in protein visualization, the dye is also used to determine the concentration of protein in the sample and in forensic labs to identify the sex of the given fingerprint.

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