The BCA assay (bicinchoninic acid assay) developed by Paul K. Smith et al. has become one of the most widely used protein assays since its introduction in 1985. The advantages offered by the assay help to explain the popularity and widespread use over other protein assays. The advantages of the BCA include:
- Compatible with detergent concentrations up to 5%
- Offers a low variance in color change from one protein to another
- Can be applied to a wide working range from 20 to 2000 µg/mL.
While these advantages for the BCA assay make it a very popular, other assays have advantages in situations that present themselves often enough to make them very useful to keep around in addition to the BCA assay.
Firstly the BCA assay is the only one of these assays that needs a prolonged incubation time in an incubator to perform. Furthermore since the BCA assay relies on the reduction of copper for color change it is susceptible to reducing agents, like DTT and 2-mercaptoethanol, and chelating agents. In addition, the majority of assays except the biuret assay are sensitive to aromatic amino acids (tyrosine, tryptophan, and, to a lesser extent, phenyalanine), and the Bradford assay specifically is also sensitive to basic amino acids (arginine most notably, but also histidine, and lysine) in addition to aromatic amino acids. This means if these amino acids aren’t found, the assay doesn’t change color, but also that if the protein sample is rich in these amino acids these assays will return a higher than normal signal.
Biuret reagent can be used as a quick, easy, and very inexpensive alternative to the BCA assay with minimal interference from most buffers; however, it is much less sensitive than other assays. To make up for this flaw a Lowry assay can be used as it is essentially adds a second step to the biuret reaction by the addition of Folin–Ciocalteu reagent (a mixture of phosphomolybdate and phosphotungstate). This second step makes the assay much more sensitive than just the biuret reagent. Like the BCA assay both the biuret assay and the Lowry assay operate off of the reduction of cupric ions to cuprous ions and therefore neither of them are compatible with reducing reagents and chelating agents. The Lowry assay is also interfered with by numerous other agents commonly found in buffers such as EDTA, tris, detergents, ammonium sulfate, and glycine to name a few. Some commercial assays are available that employ a clean-up step making the Lowry assay compatible with reducing agents, detergents and other interfering agents.
The Bradford assay also has its advantages over the other assays the biggest being that, like the biuret assay, it is a very quick and easy to perform, but unlike the biuret assay the Bradford assay is much more sensitive. Another advantage for the Bradford assay is that since it only relies upon a direct binding to protein samples this assay is compatible with a wide range of agents that the Lowry and BCA assays aren’t incompatible with. The most notable exception to this compatibility is that detergents will cause the coomassie dye reagent to precipitate. Further disadvantages include:
- High variation when assaying two different proteins
- Higher sensitivity to basic as well as aromatic amino acids (most notably arginine)
- Requires a minimum length of protein generally must be >3000 daltons for the dye to bind
- The dye stains glass and quartz cuvettes.
Protein determination by UV absorbance is the quickest and simplest way to determine protein concentration in a solution. This assay is very often used only for quick and rough estimates of protein concentration because it can be very inaccurate if the protein sample is not pure and the molar extinction coefficient of the particular protein is not known. This method typically relies upon UV absorbance of aromatic amino acids tyrosine and tryptophan at 280 nm, but it can also be performed at 220-240 nm on pure protein samples (since many other substances absorb at this range) which corresponds to the UV absorption of peptide and carboxyl groups.
There are many great and useful options to choose from for determining protein concentration in a solution than just the BCA assay. Biuret reagent presents a quick and dirt cheap solution, but not very sensitive. The Lowry assay fixes the sensitivity issues of the biuret assay but introduces the unwelcome possibility for many interfering substances. The Bradford assay is also cheap, quick, quite sensitive, and compatible with many substances that the Lowry assay isn’t; but it has high variation and won’t work with detergents. UV absorption is probably the quickest and easiest to perform, but can be pretty inaccurate if the protein sample isn’t pure or the molar extinction coefficient isn’t known.
Related Blog Articles
- Bradford Protein Assay: Calculation of An Unknown Standard
- Colorimetric or Copper Based Protein Assays - How to Decide Which is Best
- What Are the Most Commonly Used Protein Estimation Assays? (Part 1 of 2)
- What Are the Most Commonly Used Protein Estimation Assays? (Part 2 of 2)
- Spectrophotometry and Its Application in Protein Estimation
- Protein Estimation Assays: Why Is There No Single Solution?
- How to Deal with Interfering Agents in Protein Estimation Assays
- Overcome effects of detergents & reducing agents in protein estimation
