Biotech Basics: Staining Proteins

I spy with my little eye an 50 kilodalton protein! Such a statement could mark the triumphant end of a compound discovery project, medical diagnosis, or research project. Proteins can be identified and sized using electrophoretic separation on a polyacrylamide gel. (Learn more about this method, called SDS-PAGE, here and here.) However, analysis with SDS-PAGE relies on a basic but essential final step – seeing the proteins. This is accomplished using specially developed staining solutions.

Protein staining solutions need to accomplish three things. They need to travel through the gel and find proteins. Next, they need to flag the found proteins in a way that can be visualized by the scientists. Finally, they need to ensure that the flagged proteins stay stationary during subsequent wash steps and despite the inevitable process of diffusion that begins once an electrophoresis current is turned off.

Coomassie Brilliant Blue G in Isopropanol. LHcheM, CC BY-SA 3.0 https://creativecommons.org/licenses/by-sa/3.0, via Wikimedia Commons

One dye that has proven particularly adept at the find and flag part of the mission is Coomassie Brilliant Blue or sometimes CBB. CBB was discovered at the end of the 19th century. It was originally used as a wool dye which is perhaps where it gets its somewhat unscientific and brilliant name. Then in 1964, a biochemist by the name of Fazekas de St. Groth used it to stain proteins that had been separated on cellulose acetate sheets. His method was quickly adapted to the more popular approach of SDS-PAGE separation. Today, CBB staining continues to be one of the most popular protein staining methods.

There are two subtypes of CBB staining: classical and colloidal. Classical CBB is highly sensitive and can reveal all types of proteins – even those that are small or at a very low concentration. However, it does this by using a super-clingy form of CBB that also stubbornly stays in the gel. This means that classical CBB requires extensive and aggressive destaining. This destaining takes valuable lab time and sometimes reduces the overall sensitivity of the procedure. Classic CBB’s main advantage is that it’s compatible with downstream analysis like mass spectrometry. Colloidal CBB is sometimes known as InstaStain CBB. It is used more broadly because the process takes less time, is more sensitive, and usually generates less dangerous waste.  

Below is an outline of how what is happening during both types of CBB staining.

  1. Following electrophoresis (where proteins in a sample were pushed through a gel matrix by a current and traveled at different speeds depending on their sizes and sometimes charges), a gel is washed. This is because CBB also binds to the SDS detergent used during electrophoresis which creates background. (Background = nonspecific staining that interferes with observing the targeted molecules.) Because CBB also binds to proteins in the skin and on clothing, this is also a good time to double-check that everyone involved has gloves and lab coats on!
  2. The gel is emersed in a mixture of CBB, alcohol, and acid. The CBB travels throughout the gel from areas of high concentration to areas of low concentration and when it finds a protein it binds to it. Simultaneously, the alcohol and acid are also diffusing through the gel causing the proteins to precipitate which keeps them in their specific gel positions. During this step, it’s fun to imagine CBB as a massive search and find team of molecular treasure hunters searching for protein treasure in a network of interconnected caves.
  3. CBB continues to flow from the staining solution into the gel until the gel and solution reach equilibrium. This is when the concentration of dye outside the gel is the same as the concentration of dye inside the gel. At this point, dye fills all the pores of the gel. In places where CBB is bound to a protein, it’s trapped and not moving because the protein is trapped and not moving. Everywhere else CBB is moving around but because the rate that CBB molecules enter a pore is the same as the rate that they exit a pore (equilibrium) you can’t see this movement. 
  4. The stain solution is removed and replaced with a destaining solution. This solution is usually identical to the staining solution as far as the acid and alcohol goes but has no CBB. Slowly this clear solution diffuses through the gel. As it does enough of the non-protein-bound CBB exist for the proteins in the gel to be observed as dark blue bands against a light blue background.
  5. Once a gel has been stained like this, scientists determine what proteins are present in a sample as well as critical properties of these proteins such as their molecular weight and/or charge.

Because protein staining is an essential procedure in so many research and medical labs, several other staining technologies have also been developed including silver, fluorescent, and zinc staining. (Find out about these alternative methods here.) However, CBB stains remain incredibly popular because it effectively balances time requirements, safety, and sensitivity. Check out one of our protein experiments and dive into protein separation, staining, and analysis. And lookout for our new CBB method that’s formulated to decrease stain time and optimized to work with classroom-friendly acids and alcohols, coming soon!

Results from our popular experiment Survey of Protein Diversity (Kit #150)

Title Image Credit: Larionova.marina, CC BY-SA 4.0 via Wikimedia Commons

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