Bradford Assay Calculator

The Bradford assay converts a colour change into a protein concentration. Coomassie Brilliant Blue G-250 exists in acidic solution mainly as a doubly protonated red form absorbing near 470 nm; on binding protein it is stabilized in its unprotonated blue anionic form, whose absorbance maximum sits at 595 nm. Binding is electrostatic and hydrophobic, favouring arginine residues most strongly and lysine and the aromatic residues secondarily, so the signal reports available basic and hydrophobic surface rather than mass or peptide bond count. Quantification is therefore always relative to a standard curve: absorbance at 595 nm is regressed against known concentrations of a reference protein, and an unknown is read back as concentration equals corrected absorbance minus intercept, divided by slope, multiplied by any dilution factor applied before the reading. Useful working ranges are about 1 to 25 micrograms per mL for the micro assay and roughly 100 to 1500 micrograms per mL for the standard format, with corrected absorbance kept between about 0.1 and 1.0 where the response is closest to linear. The dye-protein response is mildly non-linear across the full range, so a quadratic fit is sometimes preferred over a straight line. Three limitations dominate in practice. Protein-to-protein variation can be large, because a protein poor in arginine gives far less colour per microgram than bovine serum albumin does; detergents interfere strongly, with SDS above roughly 0.1 percent being the classic case; and colour development, complete in about five minutes, drifts over the following hour, so standards and samples must be read on the same schedule.

Protein quantification before 1976 relied mainly on the biuret reaction and on the Lowry method published by Oliver Lowry and colleagues in the Journal of Biological Chemistry in 1951, which became one of the most cited papers in the scientific literature. Lowry was sensitive but slow, taking around 40 minutes, and it was notoriously vulnerable to interference from reducing agents, chelators, and buffer components common in enzymology. The dye itself came from an older tradition entirely: Coomassie is a textile dye name dating to the late nineteenth century, and Coomassie Brilliant Blue had been used for years to stain protein bands in gels before anyone quantified with it in solution. The observation that Coomassie binding to protein shifted its absorbance maximum was reported in the mid-1970s, and Marion M. Bradford, then at the University of Georgia, developed it into a practical assay published in Analytical Biochemistry, volume 72, pages 248 to 254, in 1976. The advantages were immediate: a reading in five minutes, sensitivity into the low microgram range, and tolerance of reducing agents such as dithiothreitol and mercaptoethanol that defeated the Lowry method. The paper became one of the most cited in analytical biochemistry, and commercial kits from Bio-Rad and later Thermo Fisher standardized the reagent. Its known weaknesses, particularly detergent interference and protein-to-protein variability, later motivated the bicinchoninic acid assay described by Paul Smith and colleagues in 1985, which remains the usual alternative when detergents are present.