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BCA and Bradford protein assays

All of our allergen source materials will be delivered with a certificate of analysis, which states the protein content (mg/g). To provide a comprehensive overview, we include the protein content determined through two protein determination assays on our certificates: the BCA (bicinchoninic acid) protein assay and the Bradford protein assay.

What are the disadvantages and advantages of these two protein assays? On this page, you can find more information about the BCA and Bradford protein assay methods. If you have any questions after reading this, please feel free to reach out to us. Citeq has been contributing to the field of allergy research, both therapeutics and diagnostics, for over 25 years. We specialize in house dust mites, insects and moulds and have mainly been delivering well-defined allergen source material for research and development.

Which method is better?

There is no protein assay method that is perfectly specific to proteins or uniformly sensitive to all protein types. Therefore, the successful use of protein assays involves selecting the method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that exist.

Historically, the BCA method has been found to be more sensitive than the Bradford method. This is because the BCA method is based on protein-copper chelation and the secondary detection of the reduced copper. On the other hand, the Bradford method is based on protein-dye binding, resulting in a color shift from 465 to 595 nm. This method specifically measures the presence of basic amino acid residues such as arginine, lysine, and histidine, which contribute to the formation of the protein-dye complex.

It is worth noting that the content of arginine, lysine, and histidine in Der p 1 is low, at approximately 10%.

BCA (bicinchoninic acid) protein assay

The BCA (bicinchoninic) protein assay is a widely used method for the colorimetric detection and quantification of total protein in a solution. It is a copper-based protein assay and is also known as the Smith assay, named after its introduction by Paul K. Smith et al. in 1985. One of the main advantages of this method is its compatibility with most protein samples, including those containing up to 5% surfactants (detergents). Additionally, the BCA assay demonstrates a more uniform response to different proteins compared to the Bradford method.

The BCA assay is based on the traditional Lowry assay but employs bicinchoninic acid. The assay involves two steps. The first step is the biuret reaction, which produces a faint blue color as a result of the reduction of cupric ions to cuprous ions. The second step involves the chelation of BCA with the cuprous ions, resulting in an intense purple color. The purple-colored reaction product is formed by the chelation of two BCA molecules with one cuprous ion. This BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm as protein concentrations increase. The purple color can be measured at any wavelength between 550 nm and 570 nm with minimal loss of signal (less than 10%). BCA is sensitive and has a broad dynamic range, capable of measuring protein concentrations ranging from 0.5 μg/mL to 1.5 mg/mL. An advantage of BCA is that the reagent is fairly stable under alkaline conditions and can be included in the copper solution, allowing for a one-step procedure.

Substances that reduce copper or chelate the copper can interfere with the accuracy of protein quantitation in the BCA assay. Additionally, certain single amino acids such as cysteine, cystine, tyrosine, and tryptophan can produce color and interfere with BCA assays. The BCA assay is not compatible with reducing agents. Despite these considerations, the BCA assay offers numerous advantages over other protein determination techniques.

Advantages of the BCA protein assay

  • The colour complex is stable
  • There is less susceptibility to detergents
  • It is applicable over a broad range of protein concentrations
  • The BCA assay is highly sensitive
  • It is compatible with a wide range of ionic and non-ionic detergents, and denaturing agents
  • It is capable of providing greater protein-to-protein uniformity since it is not greatly affected by differences in protein composition.
  • It can be used to assess yields in whole cell lysates and affinity column fractions.

Disadvantages of the BCA protein assay

  • The reaction may be less sensitive to the type of amino acids present in the solution but the reaction is influenced by cysteine, tyrosine and tryptophan residues. The presence of these amino acids will produce color that may interfere with your results.
  • The presence of reducing agents, copper chelating agents, acidifiers, reducing sugars, lipids and phospholipids in the buffer can also affect the accuracy of the results.
  • The interpretation of your results depends on a standard curve from a known protein sample. Thus, you need to assay samples and known proteins simultaneously using the same temperature and incubation time to get accurate results.
  • The assay requires the preparation of a working solution from supplied reagents.
  • The assay development requires long incubations of 30 minutes up to 2 hours.

Bradford protein assay

The Bradford assay is the fastest and easiest protein assay to perform among the available methods and requires a similar amount of protein as the Lowry assay. Additionally, it is compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents. The Bradford assay provides reasonably accurate results, and samples that fall outside the standard range can be retested within minutes. It is highly recommended for general use, particularly in determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis.

The Bradford method is a dye-based assay that relies on the binding ability of Coomassie blue to proteins, causing the dye to shift from a red color to a blue color. The use of Coomassie G-250 dye as a colorimetric reagent for the detection and quantification of total protein was first described by Dr. Marion Bradford in 1976 (Bradford, 1976). In the acidic environment of the reagent, proteins bind to the Coomassie dye, resulting in a spectral shift from the reddish/brown form of the dye (with an absorbance maximum at 465 nm) to the blue form of the dye (with an absorbance maximum at 610 nm). The greatest difference between the two forms of the dye occurs at 595 nm, making it the optimal wavelength to measure the blue color of the Coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm. The Bradford assay provides quick results, as samples can be read just 5 minutes after the addition of the dye to the sample.

The Bradford assay is favored by many due to its reduced interference from various substances, with the exception of high concentrations of certain detergents, which can pose a challenge if a large amount of detergent is required to lyse cells. Protein samples commonly contain salts, solvents, buffers, preservatives, reducing agents, and metal chelating agents, which are frequently used for protein solubilization and stabilization. Other protein assays like BCA and Lowry are ineffective when such molecules, especially reducing agents, interfere with the assay. The Bradford assay offers an advantage in this regard, as it is compatible with these molecules and does not experience interference. However, it is only compatible with low concentrations of detergents. If the protein sample being assayed contains detergents in the buffer, it is suggested to use the BCA protein determination procedure. Coomassie dye-containing protein assays are generally compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents commonly found in protein samples.

Protein samples typically contain salts, solvents, buffers, preservatives, reducing agents, and metal chelating agents, which are frequently used for protein solubilization and stabilization. Other protein assays like BCA and Lowry are ineffective when such molecules, especially reducing agents, interfere with the assay. The compatibility between the Bradford assay and these molecules provides an advantage, as they do not interfere with each other.

Advantages of the Bradford protein assay

  • The biggest advantage is the speed of this method. The entire process take about a half hour. This allows you to test several samples in a short amount of time.
  • Other protein assay like BCA and Lowry are ineffective because molecules like reducing agents interfere with the assay. Using Bradford can be advantageous against these molecules because they are compatible to each other and will not interfere
  • The test uses visible light (instead of UV light) to measure the absorbance of the sample. This way you don’t need a UV spectrophotometer but you can usa a simple visible light spectrophotometer.
  • The Bradford assay is able to detect a large range of proteins, detecting amounts as small as 1 to 20 μg.
  • It is an extremely sensitive technique and also very simple: measuring the OD at 595 nm after 5 minutes of incubation.

Disadvantages of the Bradford protein assay

  • The incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. In general, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent.
  • The Bradford dye reagent is highly acidic, so proteins with poor acid-solubility cannot be assayed with this reagent.
  • Finally, Bradford reagents result in about twice as much protein-to-protein variation as copper chelation-based assay reagents.
  • The Bradford assay is linear over a short range, typically from 0 µg/mL to 2000 µg/mL, often making dilutions of a sample necessary before analysis. In making these dilutions, error in one dilution is compounded in further dilutions resulting in a linear relationship that may not always be accurate.