The Bradford Protein Assay, often associated with BSA due to its common use as a standard, is a popular method for protein quantification. While it offers many advantages, it is not without limitations or potential interferences. Understanding these limitations ensures the accurate interpretation of results.

Interferences:

1. Detergents: Some detergents can interfere with the assay, even though the Bradford assay is less sensitive to detergents than other protein assays. For example, high concentrations of sodium dodecyl sulfate (SDS) or Triton X-100 can cause issues.
2. pH Sensitivity: The Bradford reagent is acidic, and therefore the assay is sensitive to pH. Buffers or other substances that alter the pH can potentially interfere with the assay.
3. Absorbing Compounds: Compounds that absorb light at or near 595 nm can interfere with the readings. For example, hemoglobin, which gives a strong absorbance in this range, can result in falsely elevated protein estimates.
4. Color-Producing Compounds: Some compounds might react with the Coomassie dye or produce color in the presence of acid, leading to interference.
5. Reducing Agents: Some reducing agents can cause the Coomassie dye to fade or bleach, which can interfere with accurate readings.

Limitations:

1. Protein Variability: Not all proteins bind the Coomassie dye with the same affinity. This means different proteins might not produce the same color intensity even at the same concentration. This can be a limitation if comparing samples with significantly different protein compositions.
2. Precision: While the Bradford assay is generally accurate, it might be less precise than other methods, especially at very low protein concentrations.
3. Narrow Dynamic Range: The linear range for the Bradford assay (where the absorbance is directly proportional to protein concentration) is relatively narrow compared to some other methods, often between 1-20 μg/mL. This means samples might need to be diluted or concentrated to fall within this range.
4. Time-Sensitive Color Development: Even though the blue color developed in the Bradford assay is relatively stable, it’s best measured shortly after mixing for optimum consistency.
5. Sample Dilution: Some samples may require dilution to fall within the linear range of the assay, potentially introducing dilution errors or making it challenging to detect very low concentration proteins.
6. Inter-sample Variation: Different samples may contain different types of interfering substances. It’s essential to treat and measure samples in a consistent manner and be aware that some samples might require method modifications.

While the Bradford assay has these potential interferences and limitations, it remains a valuable tool for protein quantification because of its simplicity, speed, and reduced sensitivity to many common contaminants. By being aware of these factors, researchers can make informed decisions and choose the most appropriate assay for their specific needs.