You can’t load a gel, set up an enzyme assay, or normalize a western blot without knowing how much protein is in your sample. The three workhorse colorimetric assays — Bradford, BCA, and Lowry — each have specific strengths and weaknesses.
How each assay works
Bradford
Coomassie Brilliant Blue G-250 dye binds primarily to arginine and aromatic residues. Binding shifts the dye’s absorbance from 465 nm (red) to 595 nm (blue). Read at 595 nm and interpolate against a BSA standard curve.
BCA (bicinchoninic acid)
A two-step reaction: peptide bonds reduce Cu²⁺ to Cu⁺, then BCA chelates Cu⁺ to form a purple complex with peak absorbance at 562 nm. The reduction is partly contributed by cysteine, tryptophan, and tyrosine residues, making BCA more uniform across different proteins than Bradford.
Lowry
A two-step reaction combining biuret (Cu²⁺ reduction by peptide bonds) with Folin-Ciocalteu reagent reduction by aromatic amino acids. Read at 750 nm. Older and slower but still used in labs that prioritize the historical reference data.
Side-by-side comparison
| Property | Bradford | BCA | Lowry |
|---|---|---|---|
| Time | 5–15 min | 30 min at 37 °C | 40 min |
| Linear range | 1–20 µg | 20–2,000 µg/mL | 1–1,500 µg/mL |
| Detergent compatible | No | Yes | Limited |
| Reducing agent compatible | Yes | No | No |
| Protein-to-protein variation | High | Low | Moderate |
| EDTA tolerance | Good | Limited | Limited |
Choosing by sample type
Bradford works best when…
- Your sample contains reducing agents (DTT, β-mercaptoethanol)
- You need a quick answer (kinetics, fractions during a column run)
- Your samples are detergent-free
BCA works best when…
- Your sample contains detergents (SDS, Triton X-100, NP-40, RIPA)
- You need protein-to-protein consistency across diverse samples
- You’re working with serum or membrane preparations
Lowry works best when…
- You need to match historical data from older publications
- Your application requires its specific dynamic range
Interfering substances
- Detergents destroy Bradford by competing for the dye.
- Reducing agents destroy BCA by reducing Cu²⁺ before BCA can react.
- Buffer components (high salt, glycerol, sucrose) can bias all three.
- Phenol or guanidine from RNA extractions interferes with all colorimetric assays.
If your lysis buffer is incompatible, you can dilute the sample (often enough to bring interferents below threshold) or switch to a fluorescent assay (Qubit Protein) for accurate quantification.
Best practices
- Always run a standard curve with each plate — never rely on yesterday’s curve
- Use BSA, IgG, or — better — a protein matched to your sample type as standard
- Run in triplicate at minimum
- Read in the linear range — dilute high-concentration samples
For most cell lysates with detergents, BCA is the safe default. For column fractions with DTT, use Bradford. For high-throughput screens with diverse buffers, validate the assay against your specific buffer first — it’s the single best predictor of whether your protein numbers will mean anything.
