How to determine the protein concentration?

Determining protein concentration is an important preliminary step in protein analysis, as it is often necessary to do before processing protein samples for isolation, purification and down-stream analysis. To date a variety of methods, differing in sensitivity, specificity and time of completion have been developed to determine protein concentration.

The simplest method to determine protein concentration is to measure the absorbance of the protein solution at 280 nm. At this wavelength, amino acids containing aromatic acid side chains, such as phenylalanine, tryptophan and tyrosine, exhibit strong absorption. The measured absorbance is proportional to the total concentration. This method, however, is not suitable for protein mixtures. Aromatic amino acid content is not consistent among the different types of proteins thereby affecting its absorption properties. Furthermore, non-protein contamination could also affect absorption. To address these limitation, several colorimetric and fluorimetric protein determination assays have been developed, these include the Bradford Assay and the BCA assay.

The Bradford assay relies on the association of Coomassie-Brilliant Blue G-250 dye with negatively charged protein molecules under acidic conditions. This results in a shift in the absorbance from 470 nm (reddish brown) to 595 nm (blue). The absorbance of the dye-protein association at 595 nm is proportional to the protein concentration.

The bicinchoninic acid (BCA) assay involves copper chelation by proteins, followed by colorimetric detection of reduced cuprous ion. Proteins can reduce Cu²⁺ to Cu¹⁺ in an alkaline solution, which will result in a purple color formation by bicinchoninic acid. The amount of ^Cu2+ reduced is proportional to the amount of protein present in the solution.

References:

Roger L. Bertholf, PhD, DABCC, FACB, Proteins and Albumin, Laboratory Medicine, Volume 45, Issue 1, February 2014, Pages e25–e41.