Coomassie, Enhanced Coomassie, and Colloidal Coomassie: What’s the Difference?

Among all the protein staining techniques available today, most researchers around the world prefer using Coomassie dyes (also known as Coomassie brilliant blue) in visualizing electrophoresed proteins. There are a number of good reasons why they do. Here are some of them:

• They offer fast and convenient protocol. When using Coomassie stains, it would only take several minutes before the protein bands are rendered visible, with maximal staining achieved within one hour. In comparison with the silver and fluorescent staining techniques, it also involves fewer steps and uses fewer reagents.  
• They provide good sensitivity. Coomassie stains may not be as sensitive as silver and fluorescent stains but they offer nanogram level sensitivity and provide good quantitative linearity.
• They are compatible with most downstream applications. Coomassie dyes do not modify the target protein in any way and works well with protein electrophoresis gels. They are also suitable for native PAGE, SDS-PAGE, isoelectric focusing and 2D gels.  
• They cost less. It costs a lot less as compared to the other staining techniques and does not require any expensive equipment (such as a couple-charged device camera (CCD) or fluorescence scanner) for imaging. 

Coomassie Dyes: Exploring the Differences Between Variants

There are several types of Coomassie stains available – the original or classical Coomassie, the colloidal Coomassie and enhanced Coomassie dyes. While all of these variants have the same general characteristics, there are also some differences between them. Thus, you need to know about these differences to help you choose the most appropriate variant for your desired application.

Classical Coomassie Stain

The classical Coomassie stain is one of the two forms of a disulfonated triphenylmethane compound commonly used in detecting and quantitating proteins. When using this variant, the dye is dissolved in a solution containing 20% to 45% methanol and 5% to 10% acetic acid at concentrations 0.1% to 0.25%. To visualize the bands, the characteristic background staining is removed by washing the gel with methanol/acetic acid.

Traditional Coomassie staining methods typically require a long time period for staining and destaining to achieve a clear background. However, prolonged destaining periods may also reduce the staining intensity of protein bands, leading to reduced sensitivity. Despite these limitations, researchers still use this technique since aside it involves a simple protocol, does not modify the target protein and is compatible with mass spectrometry. In addition, it is also a lot cheaper.  

Colloidal Coomassie Stain

Basically, Coomassie dyes share almost the same chemical structure, save for the fact that colloidal Coomassie (G-250) has two additional methyl groups. Most protein researchers now prefer colloidal Coomassie over the classical variant since it offers higher sensitivity (4ng detection limit). It also provides higher reproducibility since it reacts with the proteins (by binding with the basic amino acids arginine, lysine and histidine) and not the gel itself. Thus, background staining is reduced and the gel bands can be visualized within a shorter period of time, even without destaining the gel.

Enhanced Coomassie Stain

Realizing the limitations of both the classical and colloidal Coomassie stains, enhanced Coomassie dyes such as RAPIDStain^TM and LabSafe GEL Blue^TM have been formulated to address the perceived gaps. Both products offer high sensitivity (4 to 8 ng) and produce sharp results within a shorter period of time. Using these dyes, you can readily visualize the protein bands within 5 to 10 minutes while maximum visibility can be achieved in an hour.   

By staining the protein molecules and not the gel itself, enhanced Coomassie stains produce highly visible bands on a crystal clear background so there is no need for destaining. If you want to further enhance the band visibility, you can easily do so by rinsing the gel with water.

Image Source: Elné