Why BCA and Bradford Assays Misread Short Peptides

Two of the most popular colorimetric tests in biology are the BCA and Bradford assays. Both are designed for proteins, and both can mislead when used on short peptides.

How BCA works

Peptide bonds, plus cysteine, tyrosine and tryptophan side chains, reduce copper(II) to copper(I). The BCA reagent then binds copper(I) and turns purple. Short peptides have few peptide bonds and often no aromatic residues, so the color is weak.

How Bradford works

The dye Coomassie Brilliant Blue binds to arginine and aromatic residues and shifts from brown to blue. Peptides under about 3 kDa respond poorly, and those with no arginine or aromatics respond hardly at all.

An example

KPV 10mg has three residues and no aromatic side chain or arginine, so Bradford would barely register it.

Better options

• UV absorbance at 214 nm
• Amino acid analysis
• HPLC with a calibration curve

Keep reading for how a calibration curve is built.

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For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure or prevent any condition. This article is educational and is not medical advice.