- Bradford Assay for protein quantification is a widely used colorimetric method based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. This rapid and sensitive technique undergoes a distinct color change from reddish-brown to blue upon protein binding, with absorbance measured at 595 nm.
- The principle relies on the dye’s interaction primarily with basic amino acids (especially arginine) and hydrophobic residues. Under acidic conditions, the dye exists in its red cationic form, but upon binding to proteins, it converts to a stable blue anionic form. This shift results in a dramatic change in the dye’s absorption maximum from 465 nm to 595 nm.
- The assay’s popularity stems from its numerous advantages: high sensitivity (1-100 µg/mL), rapid results (2-5 minutes), good reproducibility, and relative simplicity. The dye-protein complex remains stable for about an hour, providing adequate time for measurements. The method requires minimal sample volume and is considerably less susceptible to interference from common reagents compared to other protein assays.
- Sample preparation involves mixing the protein solution with the Bradford reagent in appropriate proportions. The standard protocol typically uses a ratio of 1 part sample to 30 parts reagent. Proper mixing is essential but excessive agitation should be avoided as it can lead to bubble formation, interfering with measurements.
- Standard curve preparation is crucial for accurate quantification. Bovine Serum Albumin (BSA) is commonly used as the protein standard, though other proteins can be used if they better match the sample protein. Standards should be prepared in the same buffer as the unknown samples to account for any matrix effects.
- The assay has some limitations that must be considered. The response varies between different proteins due to differences in amino acid composition, particularly the content of basic and aromatic residues. Strong detergents, organic solvents, and high salt concentrations can interfere with the assay. Additionally, the working range is relatively narrow, requiring careful sample dilution.
- Quality control measures should include technical replicates, appropriate blanks, and multiple dilutions of samples to ensure measurements fall within the linear range. The standard curve should show good linearity (R² > 0.99) and consistent slope between experiments. Regular calibration checks help maintain accuracy.
- Interference from buffer components can be minimized by using appropriate controls and dilutions. Common interfering substances include detergents (especially SDS), organic solvents, and strongly basic buffers. When these cannot be avoided, standard curves should be prepared in matching buffer conditions.
- Modified Bradford protocols have been developed for specific applications. The micro Bradford assay uses smaller volumes for increased sensitivity, while specialized versions accommodate samples containing interfering substances. These modifications maintain the basic principle while addressing specific analytical needs.
- Data analysis involves plotting the standard curve and calculating unknown concentrations through interpolation. Modern spectrophotometers often include software for automated calculations, though manual verification is recommended. Results should be reported with appropriate significant figures and error estimates.
- Storage conditions for the Bradford reagent affect assay performance. The reagent should be stored in dark bottles at room temperature and filtered if precipitation occurs. Working solutions should be equilibrated to room temperature before use, and regular quality checks help ensure reagent stability.
- Documentation should include all experimental conditions, standard curve parameters, dilution factors, and any modifications to the standard protocol. This information is essential for reproducibility and troubleshooting. Regular method validation using known protein standards helps maintain confidence in results.
- Recent developments focus on improving the assay’s compatibility with various sample types and increasing throughput through automation. Miniaturization and adaptation to microplate formats have made the Bradford assay suitable for high-throughput screening applications while maintaining its fundamental advantages.
