Standard methods for protein quantitation rely on colorimetric assays like those involving protein-copper chelation (BCA and Lowry assays) and dye-binding based detection (Bradford and 660 Assay) or ultraviolet (UV) spectroscopy.
While colorimetric assays are easy to use, they are highly sensitive to sample components (detergents and reducing agents), protein composition and structure, and dye-binding properties. In these assays, protein concentration is determined by comparing signals from samples of unknown concentration to signals from reference standards (composed of common proteins such as bovine serum albumin), which are prepared for every measurement. Standard curve determinations differ considerably from assay to assay, affecting measurement reproducibility.
The BCA assay involves a two-step chemical reaction requiring incubation, and delivers results that can vary due to the kinetic rate of the reaction. In addition, phospholipids, chelating agents, reducing agents, and certain nonionic, oxidizing detergents can negatively impact the assay signal. The Bradford assay relies on binding of Coomassie Brilliant Blue G250 to basic amino acids, particularly arginine.
As a result, the assay outcome depends on the number of basic amino acid residues in the analyzed protein, which can vary greatly between proteins. Additionally, because the assay requires standard curve generation for every experiment, the proportion of basic amino acid residues in the protein standard needs to be similar to that of the protein being assayed.
UV spectroscopy-based quantification methods rely on the absorbance at 280 nm by a protein’s aromatic amino acids, predominantly tryptophan and tyrosine. Therefore, the absorbance at 280 nm of different proteins can vary widely (greater than twofold difference between extinction coefficients of albumin and immunoglobulin G). Additionally, proteins that do not contain aromatic amino acids, such as Protein A, cannot be quantified based on 280 nm absorbance.
Amino acid analysis delivers possibly the most accurate protein quantitation. The procedure consists of several steps—hydrolysis, derivatization, separation, and detection followed by quantitation. The results can be greatly influenced by variability in the inherent liability of a given sequence to hydrolysis, derivatization conditions, or sample contaminants (mainly presence of nonvolatile amines like Tris or glycine). Additionally, this method is expensive and may take time to obtain results if samples are sent to a third party for analysis.