User:Tghops/Bicinchoninic acid assay

The bicinchoninic acid assay( also known as the BCA assay or Smith assay) is a biochemical assay for determining the concentration of protein in a solution. Paul Smith and his colleagues first introduced this method in 1985. The BCA is a modification to the Lowry protein assay, which both rely on the biuret reaction. The BCA assay is a colorimetric detection method similar to the Bradford assay.

Reaction Mechanism
The first step of the assay is a reaction involving the chelation of Cu+2 (often from copper sulfate) with polypeptides (including proteins) under alkaline conditions. This leads to the generation of Cu+1 ions proportionally to the amount of protein present. Next the Cu+1 ion is incubated with bicinchoninic acid at an elevated temperature(37°C-60°C) for 30-60 minutes. The Cu+1 ion chelates with two molecules of bicinchoninic acid to produce a purple solution that absorbs strongly at a wavelength of 562 nm. The absorbance of the solution can be used to quantify the concentration of the solution by comparison with protein solutions of known concentrations. There are a number of things that influence the sensitivity of the assay. This assay does not have a specific end point and thus the incubation duration and temperature can determine the final absorbance. Increased incubation temperatures have been shown to increase the sensitivity of the reaction. The rate of the reaction is partially determined by the presence and arrangement of the amino acids cysteine, cystine, tyrosine or tryptophan in the protein. These residues are known to reduce Cu+2, therefore high abundance of these residues can result in greater concentrations of the colored species. It has been shown that interactions with the protein backbone also contribute to the reduction of Cu+2. Differences in protein structure and sequence have a strong influence on the rate of the reaction and the amount of colored species produced. Since the rate varies amongst proteins, the assay is not appropriate for determining absolute concentration. Commercially available kits are very commonly employed due to the ease of use. The formulations of solutions included in these kits are generally proprietary information and the specific reactants may change depending on the kit used. Differences between kits can influence the reactivity and amount of colorimetric species generated. Since reactivity based on the protein being tested and the method being used, it is important to select an appropriate standard and consistent method to achieve accurate and precise results.

Advantages of the BCA assay
The BCA assay
 * has very good sensitivity and a broad range of detectability(.5μg/ml
 * 2,000μg/ml) compared to a typical Lowry assay(10μg/ml-1,000μg/ml).
 * shows less protein to protein variability than the Bradford assay.
 * has an active reagent that is stable for up to a week, much longer than the unstable reactants used in the Lowry assay.
 * is easy to perform, requiring only the addition of the active reagent and then an incubation period.
 * can detect much smaller peptide chains than the Bradford assay; the BCA assay is compatible with polypeptides as small as 3 amino acids whereas the Bradford assay requires proteins of at least 3,000 Da to be effective.

A unique advantage of the BCA assay is that it is compatible with higher concentrations of surfactants/detergents(5%) than the Bradford or Lowry assay. This is useful because surfacants are often used in protein sample preparation (usually to lyse the cell).

Disadvantages of the BCA assay
One major disadvantage of the BCA assay is that it requires elevated temperatures to produce the colored species; therefore certain proteins may not be compatible due to thermal degradation at the higher temperatures. There are also a number of interferents that can disrupt the test including acidifiers, copper chelating agents, EDTA, lipids and phospholipids, reducing agents and reducing sugars. Chelating agents can result in low absorbance readings by preventing the reduction of Cu+2. Conversely reducing agents will give high absorbance readings because they will generate Cu+1 beyond what would be expected from the protein alone. Acidifiers and lipids have also been shown to give result in high absorbance values. It has been shown that the introduction of sodium dodecyl sulfate (a detergent) may eliminate interference of lipids. While these interferents hinder the quantification of total protein concentration, the side reactions can be used to quantify other data. For example, it has been suggested that the reactivity of the BCA assay with sugars could be used to determine reducing sugar concentrations, in the absence of proteins. Furthermore the interference caused by ascorbic acid can be used to determine how effectively desalted a protein is.

Advancements
Studies have reported modifications of the BCA protein assay using a microwave oven to achieve incubation times as low as twenty seconds. This significantly cuts down the time needed to run the assay and could potentially lead to much higher throughput for testing. This test has also been modified to fit microtiter plate format, which allows for testing of samples in the 96-well plate. This allows for a large number of samples to be tested at the same time.

Reference
Wiechelman, K., Braun, R. and Fitzpatrick, J. (1988). "Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation". Anal. Biochem. 175: 231–7. doi:10.1016/0003-2697(88)90383-1.

Stoscheck, CM. (1990). "Quantitation of Protein". Methods in Enzymology 182: 50–69. doi:10.1016/0076-6879(90)82008-P.