A280 vs BCA vs Bradford: Which Is Right?

Your purified protein reads 1.8 mg/mL by A280, 1.1 mg/mL by BCA, and 0.6 mg/mL by Bradford. Which number belongs in the assay plan?

Do not average the three values. They are different measurement systems with different assumptions. Disagreement is a clue about the sample, the blank, the calibration, or the chemistry. The useful result is the concentration supported by a method that is appropriate for this protein in this buffer.

The example values above are hypothetical. Their ordering does not identify a contaminant by itself. Start by making sure all three measurements refer to the same aliquot, dilution, concentration units, and definition of “protein.”

What each assay measures

The original Bradford assay and BCA assay describe distinct chemistries. Neither turns an unknown sample into a composition-independent measurement of your target protein. A BSA calibration is a response reference; your protein need not respond exactly like BSA.

For a highly purified protein with a reliable extinction coefficient and a clean spectrum, A280 is often convenient. For a mixture or a protein with weak absorbance at 280 nm, a compatible colorimetric assay may be more useful. The choice depends on the sample rather than a universal ranking of assays.

Figure 1. A280, BCA and Bradford use different optical signals and assumptions. Their readouts are not interchangeable simply because each is reported as protein concentration. Conceptual illustration.

Step 1: Separate total protein from target protein

All three methods can include contributions from other proteins. A preparation that is half target protein does not become a target-specific measurement because the instrument reports mg/mL.

Check sample composition with an appropriate identity and purity method. An SDS-PAGE band pattern can reveal major contaminants, but staining intensity is not automatically an accurate mass fraction. Different proteins stain differently, and overlapping bands can hide impurities.

This distinction matters when normalizing enzyme activity or calculating ligand-to-target ratios. “Total soluble protein loaded” and “purified target concentration” are different quantities. Label your result accordingly rather than letting a convenient concentration number imply purity.

If the sample is visibly turbid, investigate particulate material before trusting an absorbance reading. Clarification may improve measurement, but it also changes what remains in the sample. Record any centrifugation or filtration step and preserve material if you need to quantify the loss.

Step 2: Rebuild the blank

Use the actual sample buffer, including relevant additives. A blank made from a buffer recipe may miss carryover from elution, residual nucleic acids, or a different reducing-agent concentration. When possible, use an appropriate matched buffer fraction or prepare a blank that reproduces the final matrix.

For membrane proteins, distinguish protein signal from contributions associated with detergent, lipid, or other formulation components. Compatibility limits depend on the exact assay and concentration. Check the manufacturer's current manual for the formulation you are using; do not apply a limit from a different Bradford or BCA product.

Thermo Fisher's Coomassie Bradford assay instructions, for example, describe that product's operating conditions and interferences. Reducing agents can interfere with copper chemistry, while detergents can disturb dye-based assays. A supposedly compatible buffer should still be checked in your sample matrix.

Step 3: Audit the A280 calculation

The Beer–Lambert relationship is:

c = A280 / (ε × l)

Here, c is molar concentration when ε is in M⁻¹ cm⁻¹ and path length l is in cm. To convert mol/L to mg/mL, multiply by molecular weight in g/mol; those mass-concentration units have the same numerical conversion as g/L to mg/mL.

For a hypothetical protein with ε = 50,000 M⁻¹ cm⁻¹ and molecular weight = 40,000 g/mol, a blank-corrected A280 of 0.50 at a 1 cm path gives 10 µM, or 0.40 mg/mL. Apply the sample dilution factor afterward. Check whether a microvolume instrument already reports absorbance normalized to a 1 cm path; applying a second path correction would be an error.

Use the actual construct's sequence, including retained tags and cleavage remnants. Check whether the coefficient assumes oxidized cysteines. The work of Pace and colleagues provides a foundation for calculating and measuring protein absorbance coefficients.

Inspect the spectrum rather than only its A280 value. Nucleic acids, cofactors, and scattering can complicate interpretation. A260/A280 can flag a problem, but a ratio alone does not identify or accurately subtract every contaminant. See our guide to DNA and RNA co-purification.

Step 4: Run a dilution series that can reveal interference

Measure several dilutions within the validated working range of each method. Back-calculate every result to the original sample concentration. The corrected values should be reasonably consistent within the method's uncertainty.

If the apparent concentration changes with dilution, investigate a nonlinear response, matrix interference, or a measurement near the detection limit. Remember that diluting with water changes salt, detergent, and other additives as well as protein. Use an appropriate diluent and record the final matrix in each well.

For colorimetric assays, include a complete calibration curve in the relevant matrix where practical, replicate blanks, and sample replicates. Keep incubation time, temperature, and order of addition consistent. A high-quality fitted curve does not rescue standards that experienced a different buffer from the sample.

A spike-recovery test can help reveal a matrix effect: add a known amount of standard to a sample aliquot and assess the incremental response. However, satisfactory recovery of BSA does not prove that the target protein has BSA's response factor. It tests one part of the problem.

Figure 2. Dilution and a suitable blank help reveal whether a concentration measurement behaves consistently. Color intensity here is illustrative and does not represent measured concentrations.

Step 5: Choose the measurement you can defend

Consider a hypothetical purified enzyme for which A280 remains consistent across dilutions, the spectrum is clean, and the construct-specific coefficient is known. Bradford remains lower even with matrix-matched standards. A difference in dye response may explain the discrepancy; averaging the methods would obscure it.

Now consider a membrane-protein preparation with substantial background and unknown co-purifying material. A280 may be a poor target-concentration estimate, even if its technical replicates agree closely. Precision is not the same as accuracy. A compatible, validated colorimetric assay may better estimate total protein, while target-specific quantification requires additional evidence.

For decisions where absolute concentration is critical, an independently characterized reference or an orthogonal quantitative method may be warranted. Do not call one routine assay “ground truth” simply because it produces the expected answer.

Record enough information to compare future preparations

Save the method and kit, standard identity, buffer, dilution factors, raw readings, calibration range, blank, extinction coefficient when relevant, and replicate variation. State whether the value describes total protein or the target. Report sensible significant figures rather than copying every digit from the instrument.

This record is especially useful when comparing constructs. A change in tag, sequence, purity, or formulation can alter the measurement response and create an apparent improvement in yield. In a workflow using Orbion Bench for construct planning, keep concentration measurements tied to the exact construct and experimental record so downstream comparisons remain interpretable.

Common questions

Is A280 always better for purified proteins?

No. It is attractive when the coefficient, purity, and spectrum support it. Weak intrinsic absorbance, chromophoric cofactors, nucleic acids, or scattering can change that assessment.

Can I use one conversion factor between Bradford and BCA?

Only if you have validated it for the defined protein, matrix, and concentration range. A factor obtained for one preparation should not be assumed to apply to another protein or buffer.

What should I put in my methods section?

Report the measurement method, calibration or extinction coefficient, and relevant sample conditions. If methods disagreed, record the investigation and why you selected the final value. That explanation is more reproducible than a concentration number alone.