BCA test
BCA Protein Assay
The BCA (Bicinchoninic Acid) assay is a two-step colorimetric method used to quantify total protein in a sample. This method utilizes the biuret reaction, in which cupric ions (Cu²⁺) are reduced to cuprous ions (Cu¹⁺) by proteins in an alkaline environment. The amino acid backbone forms a color-chelate complex with copper ions, enabling highly sensitive and selective detection of cuprous ion cations. The amount of reduced cupric ions is proportional to the protein concentration, indicated by a color change of the sample solution from blue to purple, which can be measured spectrophotometrically at 562 nm using devices such as spectrophotometers, NanoDrop, and microplate readers. Specifically, the concentration of protein in the solution is proportional to the absorbance measured by these devices, allowing for estimation against a known protein standard, such as bovine serum albumin (BSA).
In the BCA method, certain interferences can occur, some of which are constructive while others may disrupt the assay process. Constructive interferences include ionic and non-ionic detergents and chelators (e.g., NP-40, Emulgen, HEPES, DTT, Triton X-100, Urea, Guanidine HCl, Sodium Acetate at pH 5.5). On the other hand, interferences that may disrupt the assay include:
Certain reagents, such as ethylenediaminetetraacetic acid (EDTA), which can interfere with the BCA assay by reducing sugars and lipids.
The presence of modified chemical residues in proteins.
To minimize the effects of disruptive interferences, several strategies can be employed:
Reducing interfering substances through dialysis.
Gel filtration.
If the protein concentration is sufficiently high, diluting the sample can reduce the effect of interference.
Overall, the BCA method is considered advantageous, as it can detect proteins at low concentrations of 5 µg/mL; in other words, it is highly sensitive and can accommodate a broad range of protein concentrations.
Lowry Method
The Lowry method is employed for the quantitative determination of total protein in blood serum. Similar to other colorimetric tests, it requires a standard curve to be established prior to measuring the protein concentrations in samples.
In brief, the Lowry method consists of two reaction stages. In the first stage, the nitrogen atoms in the peptide bonds of proteins react with cupric sulfate (CuSO₄) in an alkaline solution, resulting in the reduction of Cu²⁺ to Cu⁺ and the formation of a purple-violet protein-copper complex. In the second stage, the addition of Folin reagent and subsequent reactions result in the solution turning blue, which can be read spectrophotometrically within the wavelength range of 650 to 750 nm using spectrophotometers, NanoDrop, or microplate readers.
The reagents and standards used in this method are typically available commercially in ready-to-use protein concentration assay kits (Lowry method).
Note: Since the NanoDrop can measure higher protein concentrations than conventional cuvette-based spectrophotometers, you may need standards with higher concentrations than those provided in the commercial kit instructions.
This method requires more time compared to others; however, increasing the temperature can slightly reduce this time. Advantages of the method include its accuracy and sensitivity. Compounds commonly used in buffers for protein preparation (e.g., detergents, carbohydrates, glycerol, Tris, EDTA) can interfere with the Lowry assay and form precipitates.
Bradford Method
The Bradford protein assay is a technique developed by Bradford (1976) for measuring total protein concentration in a sample. It is primarily used for measuring protein concentrations in cellular fractions and assessing protein concentrations in electrophoresis gels. The principle of this method is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. 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. Notably, the binding of Coomassie blue to arginine is significant, as the amount of bound dye depends on the content of basic amino acids in the protein. Unlike the BCA method, reducing agents (e.g., DTT and beta-mercaptoethanol) and metal chelators (e.g., EDTA, EGTA) do not interfere at low concentrations. However, the presence of SDS, even at low concentrations, can disrupt the binding of protein to the dye. This technique is also less sensitive to disruptive factors compared to the Lowry method.
Advantages of the Bradford Method:
It is faster and easier than other methods.
Measurements are performed at room temperature, requiring no specialized equipment.
The preparation of reagents is straightforward, and the resulting color is stable and develops quickly.
It is compatible with most salts, solvents, buffers, thiols, reducing agents, and chelating agents in protein samples.
The method has been automated due to its simplicity and low volume of solutions required.
Pierce Method
The total protein quantification is a common measurement in laboratories for life sciences, biopharmaceuticals, and food and beverage industries. Some of the most commonly used methods for determining total protein include colorimetric assays, such as the Coomassie-based Bradford assay. Although the Bradford method is widely used, it has several drawbacks. Many substances, including certain detergents, flavonoids, and protein buffers, have been known to interfere with the colorimetric properties of this method. Additionally, the linearity of the Bradford assay is limited both in quality and range. The broad compatibility with materials and improved linearity of the Pierce 660 nm assay, combined with its straightforward single-reagent format, make it a more accurate and convenient method for many routine applications.
The Pierce 660 nm protein assay is a rapid, ready-to-use colorimetric method for quantifying total protein. This method is repeatable, faster, and more linear than the Bradford method. Furthermore, the Pierce 660 nm protein assay is compatible with high concentrations of common reagents, such as detergents and reducing agents. Similar to the Bradford method, protein concentration is estimated by referencing a series of standard protein dilutions assessed alongside unknown samples. Each total protein assay exhibits varying responses to different proteins. These differences are related to variations in amino acid sequences, isoelectric points, structure, and the presence of specific side chains or artificial groups that can significantly alter the protein's color response. Therefore, selecting an appropriate reference protein is key to achieving high-quality results. The ideal protein for use as a reference standard in any protein assay is a purified preparation of the same protein to be measured in the sample. In the absence of such a reference protein, a substitute protein that produces a similar color response to the sample protein may be used.
Common substitutes for reference proteins include bovine serum albumin (BSA) and bovine gamma globulin (BGG). When the sample predominantly contains albumin or the sample protein exhibits a color response similar to that of BSA, BSA serves as a suitable standard. For color responses from purified antibody samples and most non-serum protein mixtures (such as cell lysates), BGG is an appropriate standard protein.