The Bradford (or BSA) assay is a colorimetric protein assay based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 when it binds to proteins, primarily the arginine, lysine, and histidine residues. This shift is utilized to determine protein concentration by measuring the absorbance of a sample at a specific wavelength (595 nm). The changes in absorbance and their relation to protein concentration are as follows:

1. Dye Transition:

In the absence of protein, the Coomassie Brilliant Blue G-250 dye exists predominantly in a brownish-green neutral form, which has a maximum absorbance around 465 nm. When the dye binds to protein, it transitions to a stable anionic blue form with a maximum absorbance at 595 nm.

2. Proportional Absorbance Increase:

The intensity of the blue color (hence the absorbance at 595 nm) is proportional to the amount of protein present. As more protein is present to bind the dye, a greater proportion of the dye transitions to its blue form, resulting in an increased absorbance at 595 nm.

3. Standard Curve:

To relate the absorbance changes to actual protein concentrations:

• A set of protein standards (often BSA or another protein) at known concentrations is prepared and treated with the Bradford reagent.
• The absorbance at 595 nm for each standard concentration is measured.
• These values are used to plot a standard curve, with protein concentration on the x-axis and absorbance on the y-axis. This curve is typically linear within the working range of the assay.

4. Determination of Unknown Concentrations:

Once the standard curve is established:

• An unknown protein sample is treated with the Bradford reagent.
• Its absorbance at 595 nm is measured.
• Using the standard curve, the absorbance value of the unknown sample can be correlated to a protein concentration.

5. Advantages:

The direct relationship between absorbance changes and protein concentration allows for:

• Quick determination of protein concentrations.
• The ability to analyze multiple samples simultaneously.
• A broad range of measurable protein concentrations, typically from 1 to 20 μg/ml.

6. Considerations:

It’s worth noting that while the Bradford assay is useful and widely adopted, some factors can affect its accuracy:

• The assay is dependent on the specific amino acids in the protein, so different proteins might not bind the dye with the same efficiency.
• The presence of substances that absorb at or near 595 nm can interfere with the assay.
• Strong acids or bases, high concentrations of detergents, and certain reducing agents can also interfere with the dye’s ability to bind to proteins or with the stability of the dye’s color.

Despite these considerations, when used appropriately and with the right controls, the Bradford assay remains a robust and reliable method for protein quantification in many laboratory settings.