The bicinchroninic acid (BCA) assay, also known as the Smith assay, is a biochemical assay used to determine the total concentration of protein in a solution. Due to its ability to provide accurate determination of protein concentration and its compatibility with most protein sample types, protein laboratories around the world prefer the BCA assay over any other detergent-compatible assays.
The Many Advantages of BCA Protein Assay
The BCA assay’s popularity over other methods can also be attributed to a number of factors which include the following:
- The BCA assay is highly sensitive and has a linear range of 20-2,000µg/ml.
- It is compatible with a wide range or ionic and non-ionic detergents, and denaturing agents such as urea and guanidinium chloride.
- It is similar to the Lowry assay but since it is more stable under alkaline conditions, it can be carried out as a one-step process (the Lowry assay is a two-step process), making it four times easier and faster than the classical Lowry method.
- It is useful in monitoring protein contamination in industrial applications.
- 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.
- It is suitable for quantifying protein solutions in 1 ml assays or micro-wells.
Uncovering the Limitations of the BCA Assay
The BCA assay is not perfect. Here are some limitations that you may need to consider.
- 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.
Alternatives to the BCA Assay
Since there is no protein assay that is specific and/or uniformly sensitive to all protein types, you need to be able to choose the most compatible assay for your sample, choose an appropriate standard and deal with the limitations of your chosen assay. To help you along these lines, here are some great alternatives for the BCA assay that you may want to consider.
Lowry protein assay. Due to its sensitivity, precision and ease of use, many protein researchers use the Lowry assay in quantifying soluble proteins in a solution. The Lowry assay is very similar to the BCA assay save for the fact that it is based on two chemical reactions (the reaction of alkaline cupric sulphate in the presence of tartrate to form a tetradentate copper complex, and its subsequent reduction using Folin’s reagent to produce an intense blue color). When using this method, the effects of interfering agents such as Tris, EDTA, ammonium sulfate, sucrose and citrate can be minimized by using a precipitation method involving TCA and DOC.
Biuret protein assay. The Biuret assay involves a single incubation (20 minutes) and has fewer interfering agents as compared to the Lowry assay.
Bradford protein assay. The Bradford assay is ideal for general use, particularly for assessing protein concentrations for gel electrophoresis and determining protein content of cell fractions. It uses about the same amount of protein as the Lowry assay, gives fairly accurate results, and is the fastest and easiest to perform among the protein assays. It is also compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents.
Amido black protein assay. While it may not be as accurate as the other protein assays, Amido black micro-protein assay can be used to measure protein concentrations using trace amounts of proteins. It also involves an easy and fast protocol.
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