IR-based protein quantitation surpasses colorimetric assays and is independent of detergents, reducing agents, & analysis time

FTIR protein quantitation outperforms colormetric assays
IR-based quantitation (A) provides accurate and precise results, even in presence of detergent (SDS) and reducing agent (DTT). Using the Direct Detect™ IR-based quantitation system, calculated concentrations for the BSA samples matched the prediluted standards (A). In comparison, the Coomassie Plus (Bradford) assay provided calculated concentrations that differed greatly in the presence of 1%SDS (B, not shown here), and the MicroBCA assay could not provide data in the presence of 50 mM DTT. (C not shown here). (click to enlarge)
We compared IR-based protein quantitation using the Direct Detect® quantitation system to Bradford and bicinchoninic acid (BCA) colorimetric assays and showed that IR-based quantitation provides accurate results, even in the presence of detergent or reducing agent. Also, unlike colorimetric assays, the protein concentration obtained from IR-based analysis is unchanged regardless of the time delay between assay and data acquisition.

Protein quantitation using colorimetric assays can be inaccurate

The most common colorimetric assays for protein quantitation involve protein-copper chelation (BCA and Lowry assays) and dye-binding based detection (Bradford and “660” assays). While these assays are easy to use, disadvantages include the large variation in the binding efficiency to different proteins, reproducibility, and sensitivity to sample contaminants. Intrinsic protein characteristics that can affect concentration estimates in colorimetric assays include amino acid content, post-translational modifications, and protein secondary and tertiary structure. In one study, colorimetric assays gave results up to 60% different from values derived from amino acid analysis1.

Bradford assay: factors causing its inaccuracy

The Bradford assay relies on binding of Coomassie® Brilliant Blue G250 to basic amino acids, particularly arginine, and its absorbance shifts from 465 nm to ~595 nm upon binding. As a result, Bradford assay response depends on the number of basic amino acid residues in the protein. If the protein being assayed does not have a similar proportion of basic residues to the protein used for the standard curve, accuracy will be compromised. Also, there may be a large variation in assay response between different preparations of the Bradford reagent. It has been shown that the absorbance maximum of the dye-protein complex varies between 595 nm and 620 nm, depending on the dye source. Bradford assay response is affected by detergents (such as those used to solubilize membrane proteins). Finally, the Bradford assay is nonlinear at the higher end of the recommended protein concentration range, making data analysis challenging and error-prone.

BCA assay: factors causing its inaccuracy

The BCA assay involves a two-step chemical reaction. The first step requires protein binding to Cu2+, which is reduced to Cu1+ by cysteine, tyrosine, tryptophan and peptide bonds. In the second step, BCA chelates Cu1+ to form a purple complex that absorbs light at 565 nm. Because it is so sensitive to the amino acid composition of the protein, the BCA assay, like the Bradford assay, requires a standard curve for each experiment in which the protein standard has comparable amino acid composition to the protein being measured. For maximum sensitivity, the BCA assay requires heating, and assay signal can change depending on the length of incubation. The BCA assay is compatible with samples containing up to 5% ionic detergents; however, phospholipids, chelating agents, reducing agents, and certain nonionic, oxidizing detergents can affect the assay signal.