Coomassie brilliant blue method

The Coomassie Brilliant Blue method, also known as the Bradford assay, was developed in 1976 by Marion G. Bradford. This technique is widely used for protein quantification due to its simplicity and high sensitivity. The method relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins under acidic conditions. When the dye binds to proteins, it undergoes a color change from brownish-black to blue, and its maximum absorbance shifts from 465 nm to 595 nm. This shift makes it easy to measure the protein concentration using a spectrophotometer at 595 nm. The dye primarily interacts with basic amino acids such as arginine and aromatic residues like tryptophan and tyrosine. The intensity of the color produced is directly proportional to the amount of protein present, allowing for accurate quantification. One of the key advantages of this method is its high sensitivity—approximately four times more sensitive than the Lowry method. It can detect as little as 1 µg of protein, making it ideal for low-concentration samples. Another benefit is the speed and ease of the procedure. Only one reagent is required, and the entire process takes about 5 minutes. The dye-protein complex forms quickly within 2 minutes, and the color remains stable for up to an hour. This eliminates the need for strict time control, which is often necessary in other methods like the Lowry assay. Additionally, the Bradford method is less affected by common interfering substances such as ions (K⁺, Na⁺, Mg²⁺), Tris buffer, sugars, glycerol, and mercaptoethanol. However, it's not entirely free from interference. Detergents like Triton X-100, SDS, and even 0.1N NaOH can affect results. Also, the standard curve may not be perfectly linear, so Beer’s law cannot be applied directly. Instead, a standard curve must be constructed using a known protein, typically bovine serum albumin (BSA) or gamma-globulin, to ensure accuracy. Despite these limitations, the Bradford method remains one of the most popular techniques in biochemistry laboratories due to its reliability, speed, and cost-effectiveness. Its widespread use highlights its value in both research and clinical settings.

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