The Bradford Assay:
Protein Quantification

How It Works

The Bradford assay measures protein concentration by exploiting the interaction between Coomassie Brilliant Blue G-250 and protein. In its unbound state, the dye is reddish-brown and absorbs at 465 nm. When it binds to protein (specifically to arginine, tryptophan, tyrosine, histidine, and phenylalanine residues, as well as hydrophobic interactions), it shifts to its anionic blue form and absorbs at 595 nm. The increase in absorbance at 595 nm is proportional to the amount of protein present.

This is a colourimetric assay, which means you can read it with any spectrophotometer or plate reader. It is fast, cheap, and reasonably sensitive, which is why it remains one of the most widely used protein quantification methods despite being over 40 years old.

Standard Curves

The Bradford assay is not absolute. The relationship between absorbance and concentration depends on the amino acid composition of your protein, which means the response varies between proteins. To get a concentration, you need a standard curve generated with a protein of known concentration, typically BSA (bovine serum albumin). You prepare a series of BSA standards across a concentration range, measure their absorbance, fit a curve, and then interpolate your unknown sample onto that curve.

The important thing to understand is that the Bradford response is non-linear at higher concentrations because the dye becomes limiting. This means your standard curve is not a straight line across the full range, and extrapolating beyond your highest standard is unreliable. Work within the linear range of your curve, and if your sample is too concentrated, dilute it and re-read.

Common Pitfalls

Detergents interfere. SDS, Triton X-100, and other detergents commonly found in lysis buffers can bind Coomassie and produce false readings. If your lysis buffer contains detergent, either dilute it below the interference threshold or use the BCA assay instead, which is more tolerant of detergents.

Another common issue is using a single standard concentration and assuming linearity from zero. This is a quick-estimate approach and it works in a pinch, but it assumes the response is linear through the origin, which it is not. If precision matters, run a full standard curve.

What BenchCalc Does

BenchCalc includes both a Bradford assay calculator and a BCA assay calculator, each with standard curve mode and a quick estimate mode for when you need a rough number fast. It also includes a Beer-Lambert A280 calculator for direct UV protein quantification, an isoelectric point calculator, and protein molecular weight calculation from sequence.