BCA and Bradford Protein Assays
Our certificates of analysis include the protein content (mg/g) of allergen source materials, determined using two protein assays: the BCA (bicinchoninic acid) protein assay and the Bradford protein assay. These assays provide valuable information, and it is important to understand their advantages and disadvantages.
BCA Protein Assay: The BCA protein assay, also known as the Smith assay, is a widely used colorimetric method for detecting and quantifying total protein in a solution. It offers several advantages:
• Stability: The color complex formed in this assay is stable.
• Compatibility: The BCA assay is compatible with most protein samples, including those containing up to 5% surfactants (detergents).
• Broad range: It can accurately measure protein concentrations ranging from 0.5 μg/mL to 1.5 mg/mL.
• Sensitivity: The BCA assay is highly sensitive and responds uniformly to different proteins.
• Compatibility with detergents: It is compatible with a wide range of ionic and non-ionic detergents and denaturing agents.
• Protein-to-protein uniformity: The BCA assay is not significantly affected by differences in protein composition, providing greater uniformity in measurements.
• Applications: It can be used to assess yields in whole cell lysates and affinity column fractions.
However, there are some limitations to consider:
• Interference: Substances that reduce copper or chelate copper can interfere with the accuracy of protein quantitation.
• Amino acid residues: Certain amino acid residues like cysteine, tyrosine, and tryptophan can produce color and interfere with the BCA assay.
• Standard curve: The interpretation of results relies on a standard curve from a known protein sample, requiring simultaneous assay of samples and known proteins under the same conditions.
• Preparation and incubation time: The assay requires the preparation of a working solution from supplied reagents and longer incubation times (30 minutes to 2 hours).
Bradford Protein Assay: The Bradford assay is a dye-based method introduced by Dr. Marion Bradford. It offers the following advantages:
• Speed: The assay is fast, providing results within approximately 30 minutes.
• Compatibility: It is compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents.
• Visible light measurement: The absorbance of the sample is measured using visible light, eliminating the need for a UV spectrophotometer.
• Sensitivity: The Bradford assay can detect a wide range of proteins, as little as 1 to 20 μg.
• Simplicity: The OD at 595 nm is measured after only 5 minutes of incubation.
However, there are a few limitations to consider:
• Surfactant incompatibility: The Bradford assay is incompatible with surfactants at concentrations commonly used to solubilize membrane proteins, as they may cause reagent precipitation.
• Acidic reagent: The Bradford dye reagent is highly acidic, preventing the assay of proteins with poor acid solubility.
• Protein variation: The Bradford reagents result in approximately twice as much protein-to-protein variation compared to copper chelation-based assay reagents.
• Linear range: The assay is linear over a short range (typically 0 µg/mL to 2000 µg/mL), requiring dilutions for accurate analysis, which may introduce error during the dilution process.
In conclusion, there isn’t a single protein assay method that is universally superior, as each has its advantages and limitations. The choice between the BCA and Bradford assays depends on the compatibility with sample characteristics, presence of interfering substances, required sensitivity, and time constraints.