The Bradford assay is a widely used laboratory technique for determining the total protein concentration within a sample. This colorimetric method relies on a dye that changes color in response to protein presence, allowing for a straightforward and reliable estimation of protein levels. It serves as a fundamental tool in various biological and biochemical research settings where accurate protein quantification is necessary. The Bradford assay is recognized for its simplicity and sensitivity, making it a common choice for researchers.
The Underlying Science
The Bradford assay operates on the principle of Coomassie Brilliant Blue G-250 dye binding to proteins. This dye exists in different ionic forms depending on the pH of the solution. Under acidic conditions, the dye is predominantly in a reddish-brown cationic form, which has an absorbance maximum at 465 nm. When this dye binds to proteins, it undergoes a color change to blue.
This transformation occurs due to the dye’s interaction with specific amino acid residues within the protein, particularly basic and aromatic residues like arginine, lysine, and histidine. The binding process stabilizes the anionic (blue) form of the dye. This leads to a shift in the dye’s maximum absorbance from 465 nm to 595 nm. The intensity of the blue color, and thus the absorbance at 595 nm, is directly proportional to the amount of protein present in the sample. This direct relationship allows for the quantification of protein concentration by measuring the absorbance change using a spectrophotometer.
Steps for Performing the Assay
Performing a Bradford assay involves several steps. The process begins with preparing a series of protein standards with known concentrations. Bovine Serum Albumin (BSA) is a frequently used standard. These standards are typically prepared in a range such as 0.1 to 1.0 mg/mL, or 1 to 200 µg/mL, to cover the assay’s linear detection range.
Next, the Bradford reagent is added to both the prepared protein standards and the unknown protein samples. After adding the reagent, the mixture is incubated for a specific period. Following incubation, the absorbance of each sample and standard is measured at 595 nm using a spectrophotometer.
A standard curve is then generated by plotting the absorbance values of the known protein standards against their corresponding concentrations. This curve serves as a reference. The protein concentration of the unknown samples can then be determined by comparing their measured absorbance values to this standard curve. If the absorbance of an unknown sample falls outside the linear range, dilution and re-measurement are necessary to obtain an accurate reading.
Common Uses and Practical Considerations
The Bradford assay is a versatile tool in biological research and laboratory settings. It is frequently employed to determine protein yield after purification steps. The assay is also used to normalize protein loads for techniques like gel electrophoresis (SDS-PAGE) and Western blot analysis. Additionally, it is routinely used to quantify total protein in cell lysates before downstream experiments.
While widely used, there are factors that warrant consideration for accurate results. Certain substances, such as detergents and high salt concentrations, can interfere with the dye-protein binding, leading to inaccurate measurements. The assay also has a relatively narrow linear range for protein detection, typically between 1 and 200 μg/mL, meaning samples often require dilution to fall within this range. Furthermore, the binding affinity of the Coomassie dye can vary between different proteins due to their unique amino acid compositions, which can lead to protein-to-protein variability in color response. The assay is generally suitable for proteins larger than 3000 Daltons.