Biochemical assays are powerful tools in laboratory biomedical science, and they can often answer questions clearly and digitally in ways other procedures cannot. However, their accuracy is wholly dependent on the fidelity with which one can quantify the relative concentrations of proteins in solution.
To give an idea of the importance of protein quantification, the top three most-cited articles in the history of scientific publication describe techniques for measurement or preparation of protein samples. The all-time champion is the 1951 paper (with over 305,000 citations!) in which its lead author, Oliver Lowry, introduced an eponymous procedure for protein quantification. He and his colleagues adapted the biuret method, a test for the presence of proteins in alkaline solutions revealed by a purple color marking chelation of copper ions by a detection reagent, a chemical reaction first observed in the midnineteenth century. They developed and tested a new detection reagent that made the process more straightforward and reliable. This method was a vast improvement in user-friendliness and safety compared with the atavistic Kjeldahl method, which requires the surrogate derivation of protein content via emission of nitrogen liberated in ammonia from a sample exposed to heated sulfuric acid. Today’s average lab would need a much bigger fume hood to accommodate this assay.
Ease of use and reliability have been the keywords in subsequent variations on protein assays since the days of Lowry et al. However, most assays rely on similar principles—colorimetric evaluation of a solution via its linear change in absorbance at a given visible-spectrum wavelength when exposed to a protein-binding reagent. One way the Lowry method retains an advantage over contemporary procedures is that it is an “end-point” assay; in other words, the reaction between protein and detection reagent reaches a finite maximum so a single standard curve using a known protein can be used repeatedly to calibrate sample concentrations across multiple experiments.
In other commonly used assays, color continues to deepen indefinitely, and a new standard curve must be generated every time. This is achieved by measuring absorbance across a range of known concentrations in serial dilution of bovine serum albumin or immunoglobulin and calculating the concentrations of experimental samples from the equation generated by the standard curve. If it sounds tedious to repeat this procedure every time you run a protein gel, that’s because it is.
Improvements in reliability and throughput
Tedium aside, where Lowry is deficient compared with newer assays is in almost every other aspect relevant to modern biochemical techniques. Therefore, when the BCA assay replaced the Folin-Ciocalteau reagent with bicinchoninic acid, the throughput and reliability of copper-chelate protein quantification improved dramatically. The BCA assay proceeds in one easy step, making it more amenable to simultaneous measurement of many samples, in terms of both time and reagent usage. Moreover, the detection reagent is more stable in an alkaline solution, with less interference from components typical of many protein-containing cell lysates, including detergents, Tris, cations, EDTA, and reducing agents.
Similar in colorimetric principle, although quite different in chemistry, the Bradford assay employs the intrinsic property of Coomassie dye to change from red in acidic solution to blue when bound to protein. This is another straightforward, one-step procedure that allows for high throughput, with the added benefit that it develops more quickly. However, Bradford is also sensitive to the presence of detergents in protein buffers and suffers from high variability. Although the Pierce 660 assay uses a similar procedure while offering more compatibility in buffer constituents, variability is still a problem compared with biuret-based methods. Consequently, BCA has unofficially become the gold standard among biomedical laboratories for protein measurement preparatory to standard procedures such as Western blotting. Because of differences in chemistry, BCA and Bradford are mutually incompatible in the affinity of one detection reagent over another for particular amino acids. Proteins with excess cysteine, tyrosine, and tryptophan residues will skew BCA assay absorbances because of their affinity for cuprous ions, while the affinity of Coomassie dye for arginine and lysine will do the same, providing inaccurate readings.
Finally, for specialists who may be examining small peptides at low concentration or proteins associated with lipid bilayers that can interfere with standard assay reagents, there are several kits and protocols that allow reliable and reproducible measurement. One example is the CBQCA assay, designed for highly sensitive measurement of proteins in lipid solutions, which uses potassium cyanide (danger!) to stimulate reaction with amine groups, resulting in fluorescent excitation.
In addition to CBQCA, there is a seemingly limitless variety of specialty applications for protein quantification. The table below provides a summary of differences and compatibilities between the more standard assays. Although BCA is perhaps currently the most favored, there are rationales for choosing other assays based on factors such as buffers, time frames, detection limits, and wavelengths particular to spectrophotometer and microplate reader setups. What works best for the end user depends on considerations that are unique to every laboratory’s field of interest. This table uses kits provided by Thermo Fisher as a guide, although several are also offered by Sigma-Aldrich and Bio-Rad, and the more adventurous and thrifty among you can always make your own.
|Chemical Reaction||Biuret||Biuret||Protein-binding dye||Protein-binding dye||Amine-reactive dye|
|Number of reagents||2||2||1||1||3|
|Number of steps||2||1||1||1||3|
|Time of incubation||40 minutes||30 minutes||10 minutes||5 minutes||1 hour|
|Absorbance wavelength||750 nm||562 nm||595 nm||660 nm||Ex 460 nm / Em 550 nm|
|Temperature of reaction/storage||Ambient / 4°C||37°C / Ambient||Ambient / 4°C||Ambient / Ambient||Ambient / -21°C|
|Detection range||1 - 1500 μg / ml||20 - 2000 μg / ml||1- 1500 μg / ml||50 - 2000 μg / ml||10 ng - 150 μg / ml|
|Incompatibilities||Tris, cations, EDTA, detergents, reducing agents||Reducing agents, thiols, lipids||Low molecular weight samples, detergents||Compatible with more detergents and sample buffers||Tris, glycine, ammonium ions|