Bradford Assay Calculator

Calculate protein concentration from Bradford assay absorbance using BSA standard curve.

A595 of unknown sample

A595 of BSA standard 1 (lowest)

BSA concentration 1 (μg/mL) (μg/mL)

A595 of BSA standard 2 (highest)

BSA concentration 2 (μg/mL) (μg/mL)

Dilution factor of sample

Please check your inputs and try again.

Why Bradford Is Not Linear

Coomassie binds protein and shifts its absorbance maximum from 465 to 595 nm. The relationship between protein and A595 is curved, not linear, because dye becomes limiting as protein increases.

Fitting a straight line through curved data introduces systematic error at both ends. The practical fix is to keep unknowns in the middle of the standard range, or fit a quadratic.

Assay format	Working range
Standard Bradford	100–1,500 μg/mL
Micro Bradford	1–25 μg/mL
Linear-ish region	Only a fraction of the full range

The Protein-to-Protein Variation Problem

Coomassie binds primarily to arginine, and to a lesser extent lysine, histidine and aromatic residues. Response therefore depends heavily on amino acid composition.

Protein	Relative response versus BSA
BSA (standard)	1.00 — arginine-rich
IgG	~0.6 — underestimates by 40%
Gelatin	~0.3
Peptides under ~3 kDa	Little or no response

This is the assay's central limitation. Using BSA as standard for an antibody preparation understates concentration by roughly 40%, which is why IgG standards are used when quantifying antibodies. BSA is conventional only because it is cheap and stable, not because it is representative.

Interference and Assay Choice

	Bradford	BCA
Detergents	Interfere strongly	Tolerant
Reducing agents	Tolerant	Interfere strongly
Protein-to-protein variation	Large	Smaller
Time to result	5 minutes	30 minutes at 37°C

The two assays have complementary interference profiles, which is the practical basis for choosing between them. A lysate containing SDS or Triton needs BCA; one containing DTT or beta-mercaptoethanol needs Bradford.

Dye also binds to cuvettes and plastic, so readings drift if left standing. Read within 5–20 minutes and use the same vessel type for standards and samples.

Worked Examples

Example 1: Unknown A595=0.45, standards: 0.12@100μg/mL, 0.85@750μg/mL

Slope=890, C=1014×0.45-21

Result: C=394 μg/mL

Typical cell lysate protein concentration

Example 2: Diluted 5× sample: C_well=87μg/mL

Original = 87×5

Result: 435 μg/mL in undiluted lysate

Always account for dilution factor

Example 3: Standard choice changes the answer

Antibody quantified against BSA versus IgG standard

Result: Roughly 40% difference

Coomassie response depends on arginine content. The standard must resemble the sample, or the result is systematically biased.

Example 4: Choosing between assays

Lysate containing 1% Triton X-100

Result: Use BCA, not Bradford

Detergent interferes with Coomassie binding. The complementary interference profiles are the main reason to keep both assays available.

Common Mistakes

⚠️

Fitting a straight line across the whole range

The response is genuinely curved. Keep unknowns near the middle of the standard curve or fit a quadratic.

⚠️

Using BSA to quantify antibodies

Coomassie binds arginine, and IgG responds about 40% lower than BSA. Use an IgG standard for antibody preparations.

⚠️

Running Bradford on detergent-containing lysates

Detergents interfere strongly. BCA tolerates them, though it is in turn sensitive to reducing agents.

⚠️

Leaving plates to stand before reading

Dye binds to plastic and the signal drifts. Read within 5–20 minutes and keep vessel type consistent.

Frequently Asked Questions

Bradford vs BCA?

Bradford: Coomassie G-250 binds protein, A595 increases. Fast (5 min), cheap, interfering compounds: detergents (SDS>0.1%), basic buffers. BCA: Cu2+ reduced by protein, A562. More compatible with detergents, compatible with EDTA. Both require BSA standard curve.

Why BSA as standard?

BSA is inexpensive, pure, stable, well-characterized. But different proteins give different Bradford responses (dye binding depends on basic amino acids). For accurate absolute quantification of a specific protein: use that protein as standard or amino acid analysis.

Why is the Bradford curve non-linear?

Because free dye becomes limiting as protein concentration rises. The relationship flattens, so a linear fit introduces error at both ends.

Why does Bradford underestimate antibodies?

Coomassie binds mainly arginine, and IgG has less than BSA. Response is about 40% lower, so an IgG standard should be used.

When should I use BCA instead?

When the sample contains detergents. BCA tolerates them but is sensitive to reducing agents, so the two assays complement each other.

How soon should I read the plate?

Within 5–20 minutes. Dye binds to plastic over time and readings drift, so timing should be consistent between standards and samples.