Bradford protein assay

Coomassie Brilliant Blue G-250

Bradford protein assay

BSA Standard Curve

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Normalization of concentration

Bradford protein assay is a biochemical assay for determining the protein concentration in a solution. The method is based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. The dye, under acidic conditions, binds to basic (especially arginine) and aromatic amino acid residues. When the dye binds to these residues, its absorption maximum shifts from 465 nm to 595 nm, allowing the quantification of protein via spectrophotometry. The Bradford protein assay is favored for its rapidity, simplicity, and relative sensitivity.

Principle

The assay is based on the differential color change of the dye Coomassie Brilliant Blue G-250 in response to various concentrations of protein. Proteins in solution bind to the dye, resulting in a spectral shift from red to blue due to the stabilization of the anionic form of the dye. The amount of dye absorbed by the protein can be quantified by measuring the absorbance at 595 nm, which is proportional to the protein concentration in the sample.

Procedure

The procedure involves adding a known volume of the Bradford reagent to the protein sample and then measuring the absorbance at 595 nm after a certain incubation period. The protein concentration is determined by comparing the absorbance of the sample to a standard curve prepared with known concentrations of a standard protein, usually bovine serum albumin (BSA).

1. Prepare a series of standard solutions of known protein concentration. 2. Add a fixed volume of Bradford reagent to each standard and sample. 3. Mix well and incubate at room temperature for a specified time (usually 5 to 10 minutes). 4. Measure the absorbance at 595 nm using a spectrophotometer.

Advantages and Limitations

The Bradford assay is quick and can be performed in a single tube, making it less labor-intensive than other methods such as the Lowry protein assay. It also requires a relatively small volume of sample. However, the assay has some limitations. It is less accurate with very dilute protein solutions and can be affected by the presence of detergents or other substances that interfere with the dye-protein binding. Additionally, the assay has a narrow linear range, requiring samples to be within a specific concentration range for accurate measurement.

Applications

The Bradford protein assay is widely used in molecular biology, biochemistry, and other life science research to quantify protein in cell lysates, purification fractions, and other biological samples. It is particularly useful in the preparation of samples for SDS-PAGE, western blotting, and enzyme activity assays where precise determination of protein concentration is critical.

Comparison with Other Methods

The Bradford assay is often compared to other protein quantification methods such as the Lowry protein assay, bicinchoninic acid assay (BCA), and UV absorption at 280 nm. Each method has its own advantages and limitations regarding sensitivity, range, and interference from other substances in the sample.