There is no one-size-fits-all assay that can accurately and consistently determine the protein concentration in all samples. Each method has its own distinct set of advantages, disadvantages, and limitations, so it’s critical to weigh all of the variables before making a choice.
Protein laboratories routinely employ the BCA and Bradford assay methods to accommodate the greatest number of samples. Additionally, modified versions of these assays are used to accommodate specific sample requirements. What distinguishes these assays and how do you choose which one to use? Here are some important points to remember.
The BCA and Bradford assays are two methods for determining the protein concentration. These two assays operate on distinct principles and have varying degrees of accuracy. Factors to consider include protein-to-protein uniformity, sample volume, speed, and convenience, and the availability of necessary equipment (spectrophotometer, plate reader) to obtain the desired result. When selecting an appropriate assay method, it is also necessary to consider the presence of interfering substances.
Both Bradford assays and BCA protein estimation kits are available that can be used to perform in Microplates with multiple wells or in a larger volume format. So how do both Compare?
Overview to BCA and Bradford Protein Assay
What is the BCA Protein Assay?
The BCA assay, or bicinchoninic acid assay, is a biochemical assay that can be used to determine the total protein concentration in a solution. Additionally, this method is referred to as the Smith assay in honor of its inventor, Paul K. Smith. This method is comparable to the Lowry protein assay, the Bradford protein assay, and the biuret reagent. The total protein concentration in a sample can be determined by the color change of the sample solution from green to purple. This color change is proportional to the amount of protein present. Following that, we can use a colorimetric technique to determine the sample’s color intensity and protein content.
The BCA assay is similar to the Lowry assay except that it uses bicinchoninic acid, which produces a very pretty purple color that absorbs at 560 nm.
The typical BCA solution contains bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and copper(II) sulfate pentahydrate. This assay is based on two chemical reactions: the reduction of copper(II) to copper(I) via peptide bonds in protein and the chelation of two molecules of bicinchoninic acid with copper(I) ion, resulting in a purple-colored complex that absorbs light at a wavelength of 560 nm. The amount of copper(II) reduced by peptide bonds in the first reaction is proportional to the amount of protein in the sample.
The second reaction produces a stable, water-soluble BCA/copper complex which absorbs light at 562 nm and changes the color of the sample from blue to intense purple. The absorbance, which can be measured with a spectrophotometer, is directly proportional to the amount of protein present in the solution.
What is Bradford Protein Assay?
The Bradford assay is based on Coomassie blue’s ability to bind directly to protein molecules in a sample, causing the dye to change color from red to blue (absorbance at 465 nm) (absorbance at 595 nm). Apart from establishing the protein content of cell fractions, this technique is suited for determining the protein concentrations required for gel electrophoresis.
Bradford Protein Assay Principle
When protein is available, the red dye molecules’ negative sulfonic groups donate electrons to positively charged side chains such as lysine and arginine. This distorts the protein, facilitating van der Waals interactions and exposing hydrophobic residues to the dye, allowing for the formation of a stable complex with the dye in its blue anionic form. The absorption peak of the blue form is 595nm, and the optical density of the complex is linear within a certain range of protein concentrations.
Comparison of BCA and Bradford Assay
Pros and Cons of BCA Protein Assay
|Highly Sensitive||Less accurate|
|Compatible with a wide range of detergents||Relatively time-consuming|
|Greater protein-to-protein uniformity and produces a low variance in color change|
|Applicable to over a wide range of protein concentrations|
Pros and Cons of BCA Protein Assay
|Quick and easy to perform; One step||Uses a significant amount of sample.|
|More accurate compared to BCA||Adsorbs to glassware and quartz cuvettes, clothing, and skin|
|Compatible with a wide range of ionic and non-ionic detergents|
The primary distinction between BCA and Bradford assays is that BCA is a lengthy and imprecise procedure, whereas Bradford assay is a quick and accurate procedure.
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