BCA and Bradford protein assays

Protein assays

All of our allergen source materials will be delivered with a certificate of analysis which states the protein content (mg/g). For a complete picture we show the protein content of 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 more information about the BCA and Bradford protein assay methods.

Which method is better?

There isn’t a protein assay method that is either perfectly specific to proteins, or uniformly sensitive to all protein types. Therefore, successful use of protein assays involves selecting the method that is most compatible with the samples to be analysed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain.  Historically, the Lowry (BCA) method is more sensitive than the Bradford method, because the first method is based on protein-copper chelation and secondary detection of the reduced copper. Whereas the Bradford method is based on protein-dye binding and colour shift from 465 to 595 nm. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. The arginine, lysine and histidine content in Der p 1 is low (about 10%).

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BCA (bicinchoninic acid) protein assay

The BCA (bicinchoninic) protein assay is a widely used method for colorimetric detection and quantitation of total protein in a solution. The BCA protein assay is a Copper-based protein assay and is also known as the Smith assay because it was introduced by Paul K. Smith, et al., in 1985. One of the biggest benefits of this method is that the BCA protein assay is compatible with most protein samples and protein samples that contain up to 5% surfactants (detergents). In addition, the BCA Assay responds more uniformly to different proteins than the Bradford method.

The BCA assay is based on the traditional Lowry assay except bicinchoninic acid is used. The assay involves two steps. First is the biuret reaction, whose faint blue colour results from the reduction of cupric ion to cuprous ion. Second is the chelation of BCA with the cuprous ion, resulting in an intense purple colour.  The purple coloured reaction product is formed by the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The purple colour can be measured at any wavelength between 550 nm and 570 nm with minimal (less than 10%) loss of signal. BCA is sensitive and has a broad dynamic range; capable of measuring protein concentrations of 0.5 μg/mL to 1.5 mg/mL. The advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one-step procedure.

Substances that reduce copper will also produce colour in the BCA assay, thus interfering with the accuracy of the protein quantitation. Reagents that chelate the copper also interfere by reducing the amount of BCA colour produced with protein. Certain single amino acids (such as cysteine or cystine, tyrosine and tryptophan) will also produce colour and interfere in BCA assays. The BCA assay is not compatible with reducing agents. The BCA assay has many 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 is the fastest and easiest to perform among the protein assays and uses about the same amount of protein as the Lowry assay. It is also compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis.

Bradford protein assay Coomassie_Brilliant_Blue_G-250The Bradford method is a Dye-based assay and is based on the ability of Coomassie blue to bind protein causing the dye to shift from a red colour to a blue colour. 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, protein binds to the Coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465 nm) to the blue form of the dye (absorbance maximum at 610 nm). The difference between the two forms of the dye is greatest at 595 nm, so that is the optimal wavelength to measure the blue colour from the Coomassie dye-protein complex. If desired, the blue colour can be measured at any wavelength between 575 nm and 615 nm. The Bradford assay is quick – samples can be read 5 minutes after the addition of the dye to your sample.

A lot of people like the Bradford assay because fewer substances interfere with the assay with the notable exception of high concentrations of some detergents which can be a problem if you require a lot of detergent to lyse your cells. Protein samples usually contain salts, solvents, buffers, preservatives, reducing agents and metal chelating agents. These molecules are frequently used for solubilizing and stabilizing proteins. 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. However, it is only compatible with low concentrations of detergents. If the protein sample to be assayed has detergents present in the buffer, it is suggested to use the BCA protein determination procedure. The Coomassie dye-containing protein assays are compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples.

Protein samples usually contain salts, solvents, buffers, preservatives, reducing agents and metal chelating agents. These molecules are frequently used for solubilizing and stabilizing proteins. 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.

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.