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Introduction

Coomassie brilliant blue (CBB) stain is a widely used method for routine visualization of proteins separated on polyacrylamide gels. It is an organic dye that makes complexes with basic amino acids, such as lysine, histidine, tyrosine, and arginine. The stain transfers an overall negative charge to the proteins allowing their separation from the polyacrylamide gel. Different kinds of Coomassie dyes are available. They are known as Coomassie Brilliant Blue R-250 or G-250; where, the R stands for the more red, and the G for more green in the dye. In its anionic form, the CBB dye forms a stable blue complex with the proteins. The CBB staining is capable of detecting as little as 30-100 ng of protein, but the sensitivity of the method could be improved by performing it at elevated temperatures. The test could also be used with a variety of proteins and polypeptides having molecular weights greater than 3000. The assay is rapid and offers high reproducibility, excellent sensitivity, and ease of use.

Properties

  • Chemical formula; C47H50N3NaO7S2 (Sodium salt)
  • Molar mass; 03 g/mol
  • Solubility in water; Slightly soluble in cold, and soluble in hot (bright blue)
  • Solubility in ethanol; Soluble

Principle

The Coomassie Brilliant Blue G-250 dye has three forms: anionic (blue), neutral (green), and cationic (red). In an acidic environment, the red dye is converted into its blue form after binding to the protein of interest. If no protein binds to the dye, then the solution will remain brown. The CBB stain forms a strong, noncovalent complex with the carboxyl group of the protein by van der Waals force and the amino group through electrostatic interactions. During the complex formation, the red form of the Coomassie dye first donates its free electron to the ionizable groups on the protein causing a disruption of the protein’s native state and exposing its hydrophobic pockets. These pockets non-covalently bind to the non-polar components of the dye via van der Waals forces. The ionic interaction further strengthens the bond. This complex formation enables the detection of the proteins separated by the gel.

Staining protocol (Simpson., 2010)

Gel staining

  1. Place the gel in a microwave-safe plastic container and cover the gel with reagent A of the Coomassie dye solution.

Note: Use 100 mL of reagent A for minigel (8 × 10 cm or 8 × 8 cm).

  1. Heat the gel in a microwave oven until it boils (~2 min).
  2. Cool the gel for 5 minutes at room temperature with gentle shaking.
  3. Discard the used reagent A and briefly wash the gel with H2

Note: At this stage, bands containing >100 ng of proteins are identified despite the blue background.

  1. Add 100 mL of reagent B and heat the gel in the oven until the solution boils (~80 sec).
  2. Discard the hot reagent B and wash the gel with H2

Note: After this step, bands containing >50 ng of proteins could be visualized.

  1. Add 100 mL of reagent C and heat the gel until the solution reaches its boiling point (~80 sec).
  2. Discard the hot reagent C and wash the gel with H2

Note: Band containing >25 ng of proteins could be visualized after this step.

Destaining

  1. Add 100 mL of reagent D and heat the gel in the oven until the solution boils (~80 sec).
  2. Place a small piece of laboratory tissue in the solution to absorb excess dye.
  3. Cool the gel for 5 minutes at room temperature.

Note: At this stage, bands containing 5 ng of proteins or more could be detected.

  1. Repeat Steps 9-11 for two times, or shake the gel in reagent D for 15 minutes at room temperature.

Note: Some protein bands containing as little as 2.5 ng of proteins could be seen at this stage.

Applications

Quantification of proteins (Ku. et al., 2013)

The Coomassie brilliant blue stain is used in the Bradford assay, a colorimetric protein assay, to quantify the proteins separated by gel. This assay is performed by determining the absorbance shift of the Coomassie Brilliant Blue G-250. The amount of the complex present in the solution is used as an indicator for the protein concentration by measuring the intensity of the blue color after stabilization. The method is rapid, sensitive, and user-friendly for the quantification and estimation of proteins of interest.

Forensics (Brunelle., et al., 2017)

The Coomassie stain can interact with a small group of amino acids: arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan making it a useful stain for fingerprint analysis to identify the biological sex of the fingerprint originator. The study presented that the female samples have a higher absorbance as compared to the male samples when tested at similar wavelengths. The stain offers a viable and reliable method for fingerprint analysis and forensic identification.

Precautions

  • Wear protective gloves and gown while handling the Coomassie brilliant blue stain.
  • Rinse thoroughly with water in case of eye contact.
  • May cause skin irritation in case of skin exposure.
  • Keep the staining solution in a tightly-closed container and store in a dry, cool, and well-ventilated place.
  • Prevent discharge into the surroundings and if spilled wash it immediately with water.

Strengths and limitations

  • Coomassie brilliant blue stain offers high sensitivity, low background, large linear range, and ease of use for the identification of proteins separated by gel.
  • It is the most commonly used staining technique for the quantification of proteins after gel electrophoresis.
  • Protein ranging from 2.5 ng to 100 ng could be identified using the Coomassie brilliant blue stain.
  • The CBB stain has a wide array of applications in biochemistry, medical diagnosis, and paternal fingerprinting.
  • The staining and destaining may require more time and reagents.

References

  1. E. Brunelle., M. A. Le., C. Huynh., K. Wingfield., L. Halámková., J. Agudelo., & Halámek., J. (2017). Coomassie Brilliant Blue G-250 Dye: An Application for Forensic Fingerprint Analysis. Anal Chem., 89(7), 4314-4319.
  2. K. H. Ku., M. H. Lim., H. K. Oh., J. H. Yang., S. J. Jeong., & Kim., K. S. (2013). Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models. Anal Biochem, 434(1), 178-180.
  3. Simpson., J. R. (2010). Rapid coomassie blue staining of protein gels. Cold Spring Harb Protoc.
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