Protein quantification is the process of determining the concentration of protein in a solution. Accurate protein quantification is essential for many downstream applications, including SDS-PAGE, Western blotting, enzyme assays, and structural studies.

Common Protein Quantification Methods

Bradford Assay

The Bradford assay is based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. The dye shifts from brown to blue when bound to protein, with the absorbance maximum changing from 465 nm to 595 nm. The assay is quick, simple, and compatible with most buffers. It is less accurate in the presence of detergents.

BCA Assay

The bicinchoninic acid (BCA) assay relies on the reduction of Cu2+ to Cu1+ by proteins in an alkaline medium. BCA then chelates the Cu1+, forming a purple color that absorbs at 562 nm. The BCA assay is more tolerant of detergents than the Bradford assay but is less compatible with reducing agents.

Lowry Assay

The Lowry assay combines the biuret reaction (copper chelation) with the Folin-Ciocalteu reagent, which reacts with tyrosine and tryptophan residues. It produces a blue color measured at 750 nm. It is sensitive but requires careful timing and is affected by many interfering substances.

UV Absorbance (A280)

Proteins absorb UV light at 280 nm due to tryptophan and tyrosine residues. The absorbance at 280 nm provides a quick, non-destructive estimate of protein concentration. It is most accurate for pure proteins with known extinction coefficients.

Standard Curve

All colorimetric assays require a standard curve. Serial dilutions of a known protein standard, typically bovine serum albumin (BSA), are prepared and measured. The unknown sample’s absorbance is compared to the standard curve to calculate its concentration.