Choosing the Right Protein Concentration Assay: Pros and Cons of Common Techniques

Bicinchoninic Acid (BCA) assay

The BCA assay is a two-step colorimetric technique that has been used by scientists to determine protein concentrations since the mid-80s [1]. In the first step, cupric ions (Cu²⁺) react with the aromatic backbone amino acids including tyrosine, tryptophan and cystine, which reduces Cu²⁺ to Cu⁺. In the second step, Cu⁺ reacts with the BCA reagent to form a stable, purple-colored complex [2]. The absorbance of the complex at 562nm is measured with a spectrophotometer and compared it to a standard curve to calculate protein concentration.

Pros

• Versatile assay that is compatible with several detergents and chemicals occupying buffers.
• Wide dynamic range allowing quantification of proteins between 20 and 2000 µg/mL.
• Typically performed in 96-well plates, giving rise to a reasonably high throughput workflow. However it is worth noting that incubation times may be up to 1 hour.

Cons

• Relies on aromatic backbone amino acids (tyrosine, tryptophan and cystine) for the reduction of Cu²⁺ to Cu⁺. In samples containing low aromatic amino acids, concentration will be underestimated.
• Whilst well suited to some buffer components, high concentrations of reducing agents or substances known to interfere with copper (i.e. ammonia) may influence the result. Similarly, samples containing high concentrations of metal ions (Cu²⁺ or Fe³⁺) may lead to unreliable protein concentrations.
• Cannot reliably determine protein concentrations < 20 µg/mL.

Bradford assay

Developed by Marion Bradford in the 1970’s, the Bradford assay offers a simple and quick method to determine protein concentrations [3]. This one-step colorimetric assay is reliant on the interaction between “Coomassie brilliant blue” (triphenylmethane dye) and aromatic sidechain (tryptophan, tyrosine and phenylalanine) or positively charged amino acids (including lysine and arginine). Upon binding of Coomassie brilliant blue to protein, the maximum absorbance shifts from 470nm to 595nm [4]. Similarly to the BCA assay, absorbance is measured at 595nm and compared to a standard curve to give concentration.

Pros

• Requires just one step, allowing quick and easy determination of protein concentration (typically < 10 minutes).
• Unlike the BCA assay, the Bradford assay is not affected by reducing agents in the sample/buffer.

Cons

• Dependent on the sequence of aromatic or positively charged amino acids. Similarly to the BCA assay, low concentrations will reduce the binding efficiency of the dye and lead to an underestimation of protein concentration.
• Formation of the protein-dye complex is influenced by a number of detergents (including SDS, Triton X-100 and Tween), and buffer components (including Tris and EDTA) so need to be avoided.

Ultraviolet-Visible (UV-Vis) Spectrometer

Although often unreliable, another straightforward and widely used technique to determine protein concentration is measuring UV absorbance with a UV-Vis spectrometer [5]. The amino acids tyrosine and tryptophan absorb UV light at 280nm, and when the extinction coefficient of the protein is known, the Beer-Lambert law can be employed to determine protein concentration. Due to its simplicity and ease of use, UV-Vis spectrometers have found their way into the majority of protein science laboratories.

Pros

• This is a quick and easy assay that does not require any steps, reagents or incubation time.
• Protein concentrations can be determined from a low volume of sample – as little as 2 µL.

Cons

• As with the BCA and Bradford assays, a reliance on aromatic amino acids can lead to an underestimation of concentration with proteins containing fewer tryptophan and tyrosine residues.
• Lots of other molecules absorb UV light at 280nm which interferes with the measurement. This includes biomolecules such nucleic acids and other sample/buffer components such as detergents, reducing agents and phenols.
• Low sensitivity compared to other methods – measuring samples containing < 1 µg/mL of protein can be problematic.

Enzyme-linked immunosorbent assay (ELISA)

The ELISA technique was developed by Swedish scientists in the 1970’s [6]; There are several variations of ELISA, including direct, indirect, sandwich and others, each with their own advantages and disadvantages, however all share a common principle [7]. The protein of interest is captured with a specific antibody with high specificity to the target. The antibody may be pre-conjugated with an enzyme (direct/indirect ELISA) or it is added in a later step, binding directly to the target of interest (sandwich ELISA).

Following the addition of a substrate, the reaction causes a colour change, which is measured using a plate reader and compared to a standard curve, allowing the determination of protein concentration.

Pros

• Highly sensitive techniques with the capacity to measure protein concentrations in the pg/mL range.
• Due to the specificity of the antibody to the target protein, a wide range of samples and buffers can be utilized without worrying about interference.
• Performing assays in 96-well plates, as well as the ability to automate workflows, allows for high sample throughput.

Cons

• The selection, optimization and preparation of antibodies is time consuming and laborious, which can delay the start of experiments.
• The assays themselves are more time consuming, involving numerous steps and incubation times.
• ELISA is typically more expensive than other techniques. Several reagents are required, and purchasing specific antibodies can be costly.
• A further limitation, which is true for any assay utilizing protein standards and/or standard curves, is that the estimate on concentration is dependent on the quality of the protein and preparation of standards. Therefore, any inaccuracies in these factors would be reflected in the assay’s outputs.

Summary of common protein concentration assays’ pros and cons

Microfluidic Diffusional Sizing (MDS) – a quantitative and calibration-free assay to determine protein concentration and affinity

While protein concentration is undeniably a key metric for understanding the dynamics of biological systems, it alone does not provide a comprehensive view. To fully grasp the impact of a protein, both its concentration and its affinity for a target must be considered. Colorimetric assays are limited in that they cannot measure affinity, and ELISA, though useful, only provides semi-quantitative data such as titer, which cannot differentiate between a low concentration of a high-affinity protein and a high concentration of a low-affinity protein.

MDS technology, leveraging Bayesian inference, enables the simultaneous determination of both active protein concentration and affinity without the need for calibrators or a standard curve. This method overcomes the challenges associated with preparing protein standards, which can be problematic and raise concerns about the accuracy of concentrations derived from techniques reliant on standard curves. Additionally, this approach allows for the analysis of complex biological samples, such as measuring the affinity and concentration of antibodies in undiluted serum, or assessing the affinity, concentration, and copy number of membrane proteins.

The assay is simple, immobilization-free and calibration-free, taking 25 minutes to measure and using just 60 μL of serum, providing both affinity and concentration of antibody directly in clinical samples.

For further information about using MDS to quantify serum antibody concentration, visit https://fluidic.com/applications-serum-antibody-profiling/

References

[1] Smith, P.E., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Analytical biochemistry, 150(1), pp.76-85. https://doi.org/10.1016/0003-2697(85)90442-7

[2] Walker, J.M., 2009. The bicinchoninic acid (BCA) assay for protein quantitation. The protein protocols handbook, pp.11-15. https://link.springer.com/book/10.1385/1592591698

[3] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), pp.248-254. https://doi.org/10.1016/0003-2697(76)90527-3

[4] Kielkopf, C.L., Bauer, W. and Urbatsch, I.L., 2020. Bradford assay for determining protein concentration. Cold Spring Harbor Protocols, 2020(4), pp.pdb-prot102269. https://cshprotocols.cshlp.org/content/2020/4/pdb.prot102269.short

[5] Kielkopf, C.L., Bauer, W. and Urbatsch, I.L., 2020. Methods for measuring the concentrations of proteins. Cold Spring Harbor Protocols, 2020(4), pp.pdb-top102277. https://cshprotocols.cshlp.org/content/2020/4/pdb.top102277.short

[6] Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay, ELISA: III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. The Journal of Immunology, 109(1), pp.129-135. https://doi.org/10.4049/jimmunol.109.1.129

[7] Hayrapetyan, H., Tran, T., Tellez-Corrales, E. and Madiraju, C., 2023. Enzyme-linked immunosorbent assay: types and applications. ELISA: Methods and Protocols, pp.1-17. https://link.springer.com/protocol/10.1007/978-1-0716-2903-1_1