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.
Practical BCA and Bradford Assay Protocols
BCA Assay: Prepare BSA standards by serial dilution in the same buffer as the samples — a typical range is 25–2000 µg/mL (0, 25, 125, 250, 500, 750, 1000, 1500, 2000 µg/mL). Mix 25 µL of each standard or sample with 200 µL of BCA working reagent (50:1 ratio of reagent A to reagent B) in a 96-well plate. Incubate at 37°C for 30 minutes. Cool to room temperature and measure absorbance at 562 nm. Fit a quadratic or linear standard curve. The BCA assay is compatible with detergents (SDS, Triton X-100) up to 5% but is incompatible with reducing agents (DTT, β-mercaptoethanol) > 1 mM. Bradford Assay: Prepare standards in the same range (25–1000 µg/mL). Mix 10 µL of standard or sample with 200 µL of Bradford reagent (Coomassie G-250 dye in phosphoric acid and methanol). Incubate at room temperature for 5 minutes. Measure absorbance at 595 nm. The Bradford assay is rapid (2 minutes) and compatible with reducing agents and chelating agents (EDTA) but is strongly inhibited by detergents (>0.1% Triton X-100 or SDS). For both assays, include buffer-only blanks. Each standard and sample should be assayed in duplicate or triplicate. Discard readings with CV > 15%. Troubleshoot high background by diluting the sample further, switching assays (use Bradford if samples contain reducing agents, use BCA if samples contain detergents), or performing a TCA/acetone precipitation to remove interfering substances.
Real-World Application
When quantifying protein lysate from mammalian cells extracted in RIPA buffer (containing 1% Triton X-100 and 0.1% SDS), the BCA assay is preferred over Bradford. The standard curve from 25–2000 µg/mL BSA in RIPA buffer shows R² = 0.996. The unknown lysate gives A562 = 0.45, corresponding to 1250 µg/mL. After dilution, 20 µg is loaded per lane for SDS-PAGE or for analysis by capillary gel electrophoresis.
