The Bradford assay is a widely utilized and straightforward laboratory technique developed to quickly determine the total protein concentration within a solution. This colorimetric method is one of the most frequently employed procedures in biochemistry and molecular biology, providing a quantitative measurement foundational for countless experiments. Knowing the amount of protein present is often required before proceeding with subsequent analyses, such as preparing samples for gel electrophoresis or enzyme activity studies. The assay relies on a measurable change in color intensity that is directly proportional to the amount of protein present.
The Chemical Principle
The Bradford assay rests on the interaction between protein molecules and the organic compound Coomassie Brilliant Blue G-250 dye. In its acidic state, the dye exists primarily in a reddish-brown, cationic form, absorbing light maximally at 465 nanometers (nm). Introducing protein to this acidic environment triggers a rapid change in the dye’s molecular structure and electrical charge.
When the dye binds to a protein, it stabilizes into an anionic form, shifting its absorption maximum from 465 nm to 595 nm, resulting in an intense blue color. This binding occurs mainly through electrostatic interactions between the dye’s negatively charged sulfonate groups and the positively charged side chains of basic amino acid residues, primarily arginine and lysine. Hydrophobic interactions involving aromatic residues like tryptophan, tyrosine, and phenylalanine also stabilize the complex. The depth of the resulting blue color is directly correlated with the concentration of protein in the sample.
Executing the Laboratory Procedure
The execution of a Bradford assay involves precise steps to accurately measure the color change. The process begins with preparing a series of protein standards, typically using Bovine Serum Albumin (BSA) as a highly purified reference protein. These standards are carefully diluted to create a range of known concentrations that bracket the expected concentration of the unknown samples.
Next, a measured aliquot of each standard and unknown sample is combined with the Bradford reagent, which contains the Coomassie Brilliant Blue G-250 dye in an acidic solution. In a standard assay, a small volume of sample, such as 20 microliters, is often mixed with 1 milliliter of reagent in a cuvette, resulting in a sample-to-reagent ratio of about 1:50. For microplate assays, the volumes are miniaturized, but the ratio is maintained.
The components are gently mixed to avoid foaming and incubated at room temperature for 5 to 20 minutes. This allows sufficient time for the blue dye-protein complex to form and stabilize. The final step is to measure the absorbance of each sample using a spectrophotometer or plate reader set to 595 nm.
The spectrophotometer must first be “blanked” using a control solution containing only the Bradford reagent and the sample buffer, but no protein. This procedure calibrates the instrument by setting the baseline absorbance of the unreacted reddish-brown dye to zero. This ensures that the measured absorbance at 595 nm reflects only the blue-colored protein-dye complex, generating the raw data needed to quantify the unknown samples.
Interpreting Results Using the Standard Curve
The raw absorbance values must be converted into protein concentration units using a standard curve. This curve is a graphical representation created by plotting the known concentrations of the BSA standards (x-axis) against their measured absorbance values at 595 nm (y-axis). Since the relationship between concentration and absorbance follows the Beer-Lambert law within a specific concentration range, the resulting data points should form a relatively straight line.
A linear regression analysis is performed on these data points to generate an equation for the line of best fit. This equation mathematically defines the relationship between absorbance and protein concentration, serving as the calibration tool. To determine the concentration of an unknown sample, its measured absorbance value is interpolated onto this standard curve.
The unknown sample’s absorbance value is located on the y-axis, traced to the curve, and then projected down to the x-axis to reveal the corresponding protein concentration, typically expressed as mass per volume, such as micrograms per milliliter. Accurate interpretation requires the unknown sample’s absorbance to fall within the linear range of the standard curve, emphasizing the importance of proper initial sample dilution.
Advantages and Limitations
The popularity of the Bradford assay stems from several practical advantages:
- The procedure is fast, with the color-forming reaction occurring almost instantly.
- It is a sensitive assay, capable of accurately detecting protein concentrations in the microgram range.
- The reagents are relatively inexpensive.
- The final blue complex is stable for an hour or more, allowing ample time for measurement.
Despite its benefits, the Bradford assay has limitations that must be considered for accurate results. The primary drawback is the significant variability in response between different types of proteins. This variability occurs because the assay relies heavily on specific amino acid residues for binding, meaning the color yield is protein-dependent.
Proteins rich in basic residues, such as histones, produce a more intense blue color and thus appear to have a higher concentration compared to the BSA standard. Conversely, proteins lacking these residues may be underestimated.
Another challenge is the assay’s high susceptibility to interference from non-protein components frequently found in biological samples and buffers. Detergents, even at low concentrations, can interact with the dye and disrupt protein binding, leading to inaccurate readings. Other compounds, including strong alkaline buffers, can also skew results by affecting the dye’s equilibrium. Researchers often need to perform preliminary clean-up or dilution steps to minimize the impact of these interfering substances.