Article — Protein Concentration Calculator
Protein Concentration Calculator: A280, Bradford, BCA
A protein concentration calculator converts absorbance to milligrams per milliliter using either the Beer-Lambert equation (A280 method) or a Bradford / BCA standard curve. For A280, concentration equals absorbance divided by extinction coefficient times path length. For Bradford and BCA, concentration equals (sample absorbance minus intercept) divided by slope, all multiplied by the dilution factor.
Three methods dominate routine protein quantification, and each one is right for a different sample type. A280 reads native UV absorbance and works for purified protein with a known extinction coefficient. Bradford uses a Coomassie dye and gives readings in five minutes with reductant compatibility. BCA uses cupric copper reduction and offers the widest linear range, plus detergent tolerance.
What protein concentration means
Protein concentration is the mass of protein per unit volume of solution. The lab-standard unit is milligrams per milliliter (mg/mL), with micrograms per milliliter (µg/mL) for dilute samples and micromolar (µM) for molar work. Conversion between mass and molar requires the protein molecular weight: micromolar equals mg/mL times 1000 divided by MW in kDa.
Reporting concentration matters because nearly every downstream protein experiment loads a fixed mass. SDS-PAGE wants 10 to 30 µg per lane. Western blots want similar loads. Enzyme kinetics need precise micromolar starting concentrations. Without accurate concentration, every comparison across samples becomes noisy.
A280 protein concentration method
The A280 method exploits the UV absorbance of tryptophan and tyrosine residues at 280 nanometers. Both aromatic amino acids absorb strongly in the near-UV. Disulfide bonds contribute a small additional signal. The Beer-Lambert law (c = A / ε × b) converts absorbance directly to molar concentration.
A280 needs three inputs: absorbance from the spectrophotometer, extinction coefficient (computed from sequence via ExPASy ProtParam or looked up in literature), and path length (1 cm in a standard cuvette, 0.1 cm for a NanoDrop). The protein concentration calculator does the rest.
Bovine serum albumin (BSA) has an extinction coefficient of 43,824 M⁻¹·cm⁻¹ at 280 nm — meaning a 1 mg/mL BSA solution reads A280 = 0.667. The number is so well established that BSA is the universal Bradford and BCA standard.
Bradford protein concentration assay
The Bradford assay uses Coomassie Brilliant Blue G-250 dye that shifts from red (free) to blue (protein-bound). The assay takes 5 to 15 minutes at room temperature, tolerates reducing agents like DTT and beta-mercaptoethanol up to 100 mM, and reads at 595 nm. The linear range runs from 1 to 100 µg/mL.
Bradford is sensitive to detergents — Triton X-100, SDS, and CHAPS above 0.1 percent shift the binding equilibrium and inflate readings. Detergent-compatible versions (Bio-Rad DC, Pierce Coomassie Plus) reformulate the dye buffer to tolerate up to 1 percent SDS. For routine work with crude lysates in plain buffer, classic Bradford is fast and reliable.
BCA protein concentration assay
The bicinchoninic acid (BCA) assay uses copper reduction in alkaline conditions. Peptide bonds reduce cupric (Cu²⁺) to cuprous (Cu⁺) ion, which then chelates with two BCA molecules to form a violet complex absorbing at 562 nm. The reaction takes 30 minutes at 37°C or 2 hours at room temperature.
BCA covers the widest linear range of the three methods — 15 to 1,500 µg/mL — and tolerates most detergents up to 5 percent. It is the workhorse for crude membrane fractions, detergent-solubilized proteins, and lysates with mixed buffer composition. The drawback is sensitivity to reducing agents — DTT and beta-mercaptoethanol over 1 mM falsely raise readings by reducing Cu²⁺ directly.
If your sample buffer has both detergent and reductant, neither classic Bradford nor classic BCA works perfectly. Use a detergent-compatible Bradford kit, or dialyze the sample to remove reductant before BCA.
Standard curve fitting
Bradford and BCA both report concentration relative to a bovine serum albumin (BSA) standard curve. Run five to seven BSA standards (0, 25, 50, 100, 250, 500, 1000 µg/mL is typical), each in triplicate. Fit a linear regression — R² should exceed 0.99 within the linear range.
The slope (m) and y-intercept (c) feed into the protein concentration calculator. Sample concentration equals sample absorbance minus c, divided by m, then multiplied by any dilution factor applied before the assay. Discard any standard with absorbance outside 0.05 to 1.5 — outside that range, the linear assumption fails.
- R² = 0.99+ for publishable data
- standards = 5–7 BSA dilutions per curve
- replicates = 3 per standard
- linear range = absorbance 0.05 to 1.5
- NTC = no-protein blank required on every plate
- refresh = run new curve every assay batch
Dilution factors and linear range
The dilution factor is the ratio of final volume to sample volume. Add 10 µL sample to 90 µL buffer and you have a 1:10 dilution (DF = 10). After computing the diluted concentration from the standard curve or Beer-Lambert, multiply by DF to back out the original sample concentration.
Always dilute crude lysates before assay. Cell lysates routinely run 5 to 20 mg/mL — far above the linear range of Bradford (max 100 µg/mL) or BCA (max 1,500 µg/mL). A 1:50 to 1:100 dilution brings lysate into Bradford range. For BCA, 1:10 to 1:20 usually works. The protein concentration calculator applies DF automatically once you enter it.
Picking the right method
A280 if the protein is purified, you know the extinction coefficient, and the buffer has no contaminating chromophores. Bradford if you need speed and the buffer has reductants but no detergent. BCA if the buffer has detergents, the sample is dilute, or you need the widest dynamic range without dilution series. NanoDrop A280 if you have under 5 µL of pure protein and need a 30-second answer.
Bradford response to different proteins varies by up to 30 percent against BSA standards — basic proteins like histones read 2× higher than they should, and acidic proteins read low. For absolute quantification, build a standard curve from your purified protein. For relative comparisons across samples of the same protein, BSA standards are fine.
Common measurement pitfalls
Bubbles and particulates in the cuvette inflate apparent absorbance — always blank with the same buffer and look for visible specks. Wavelength drift on an uncalibrated spectrophotometer shifts readings by 5 to 10 percent — calibrate annually with a holmium oxide filter. Temperature changes alter Bradford binding equilibrium by 2 percent per degree — read all samples within 30 minutes of mixing at consistent room temperature.
A280 c = A / (ε × b)standard curve c = (A − c) / m × DFmolar to mass mg/mL = M × MW / 1000dilution c₁V₁ = c₂V₂