Bradford Assay Calculators

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The Bradford assay is a rapid colorimetric method for quantifying protein concentration based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. When the dye binds to proteins — primarily through electrostatic interactions with basic residues (Arg, Lys, His) and hydrophobic interactions — it shifts from its red/brown free form (λmax 465 nm) to a blue bound form (λmax 595 nm). The increase in absorbance at 595 nm is proportional to protein concentration and is quantified against a BSA standard curve. The Bradford assay is fast, sensitive, and widely used in biochemistry labs for routine protein quantification.

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Bradford Assay Principle

Coomassie Brilliant Blue G-250 exists in red/brown (protonated, λmax 465 nm) and blue (anionic, λmax 595 nm) forms. In acidic reagent conditions, the dye is red/brown. Protein binding stabilizes the blue anionic form, shifting absorbance to 595 nm. The amount of blue complex formed is proportional to protein concentration over the linear range of the assay (~10–1000 μg/mL for standard assay; ~1–25 μg/mL for micro Bradford).

Standard Curve Construction

Prepare BSA (bovine serum albumin) standards: 0, 125, 250, 500, 750, 1000 μg/mL (or appropriate range). Add 10 μL of each standard and unknown to 200 μL Bradford reagent in a 96-well plate (or 100 μL sample + 1 mL reagent in a cuvette). Mix, incubate 5 min at room temperature, read A595. Plot A595 vs. BSA concentration; fit a linear or polynomial curve. Interpolate unknown concentrations.

Interference and Limitations

  • Detergents: SDS, Triton X-100, and Tween cause false positive or negative readings at >0.1% concentration
  • DTT and 2-mercaptoethanol: Interfere at high concentrations
  • Protein-to-protein variation: Bradford response varies by amino acid composition — Arg-rich proteins give higher signal; Gly-rich proteins give lower signal relative to BSA
  • Cuvette staining: Dye stains plastic; use glass or disposable cuvettes

Bradford vs. BCA Assay

BCA (bicinchoninic acid) assay is more tolerant of detergents (works in up to 5% SDS) and gives more uniform protein-to-protein response. Bradford is faster (5 min vs. 30 min for BCA) and does not require heating. Choice depends on sample buffer composition and speed requirements.

Glossary

Coomassie Brilliant Blue G-250
The dye used in the Bradford assay; binds proteins to form a blue complex absorbing at 595 nm; the intensity of blue color is proportional to protein concentration.
BSA Standard Curve
A calibration curve made from known concentrations of bovine serum albumin; used to interpolate unknown protein concentrations from absorbance readings in Bradford and BCA assays.
BCA (Bicinchoninic Acid) Assay
An alternative protein quantification method based on Cu²⁺ reduction by proteins; more detergent-tolerant than Bradford; read at 562 nm after 30 min incubation at 37°C.

Frequently Asked Questions

Bradford assay is based on Coomassie Brilliant Blue G-250 binding to proteins. In acidic conditions, the free dye is red/brown (absorbs at 465 nm). When it binds protein (mainly Arg, Lys, and aromatic residues), it shifts to blue and absorbs at 595 nm. A595 is proportional to protein concentration over the linear range. Protein concentration is determined by comparing A595 to a BSA standard curve. The assay takes about 5 minutes at room temperature.

Prepare a series of BSA standards covering the linear range of the assay (typically 0–1000 μg/mL for standard, 0–25 μg/mL for micro Bradford). Add equal volumes of each standard and unknown sample to Bradford reagent, mix, incubate 5 min, and read A595. Plot A595 on the y-axis and BSA concentration on the x-axis. Fit a linear regression line. For unknowns, read their A595 values on the standard curve and calculate concentration, multiplying by any dilution factor applied before the assay.

Bradford limitations: detergents (SDS > 0.1%, Triton X-100 > 0.01%) interfere significantly — use BCA assay for detergent-containing buffers. Protein-to-protein variation exists because response depends on amino acid composition (particularly Arg content) — a protein with low Arg content will be underestimated relative to a BSA standard. Not compatible with samples containing high concentrations of free amino acids or lipids. Plastic cuvettes are stained permanently — use glass or disposable plasticware.

Use Bradford when: samples are in simple buffers without detergents; speed is important (5 min, no heating); you are processing large numbers of samples; low concentrations (micro Bradford: 1–25 μg/mL). Use BCA when: samples contain SDS (BCA works up to 5%), Triton X-100, or other detergents; you need more uniform protein-to-protein response; sample volume is very small. Both require BSA standard curves and give results relative to BSA — neither gives true absolute protein concentration.