What is the difference between Bradford, BCA, and Lowry protein assays?

Bradford detects protein via Coomassie dye binding (fast, but sensitive to detergents). BCA uses Cu²⁺ reduction followed by bicinchoninic acid chelation (compatible with detergents, but slower). Lowry combines Folin–Ciocalteu reagent with copper–protein complexes (sensitive, but has more interference issues). The choice depends on your sample buffer and throughput needs.

Why does my protein assay give inaccurate results outside the linear range?

Standard curves for colorimetric protein assays are only linear within a specific absorbance window. Below the lower limit, signal is indistinguishable from noise. Above the upper limit, the dye or reagent is saturated and additional protein no longer produces proportional signal. Always interpolate within the validated range and dilute samples that exceed it.

How do I choose the right standard curve range for protein quantitation?

Start with the manufacturer-recommended range (e.g., 0.1–1.0 mg/mL for Bradford). Prepare at least 5–6 standard points spanning the range. If your unknown falls outside the curve, dilute and re-measure rather than extrapolating beyond the last standard point.

Research workbench

Protein Assay

Enter your Bradford or BCA plate readings to get the standard curve, R² and back-calculated concentrations.

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Plate data

Concentration unit

Reagent

Wells used

Concentration (µg/mL) · absorbance A595

Sample	Mean	CV
Standards
	n/a	n/a
	n/a	n/a
	n/a	n/a
	n/a	n/a
	n/a	n/a
	n/a	n/a

Unknown sample
	n/a	n/a
	n/a	n/a

Enter the raw A595 readings from the selected wells. Leave unused wells blank. The first standard row starts at the blank.

For a full plate-reader CSV or Excel export, choose “Import reader data” and select your group’s columns. You can also click a well and paste directly.

Results

Enter readings for at least two standard rows to see the curve and sample estimates.

Assay explorer

Switch between common protein assays and compare chemistry, linear range, and buffer compatibility.

Principle

Coomassie Brilliant Blue shifts to its blue form after binding basic and aromatic residues.

Readout wavelength

595 nm

Suggested linear range

0.1 – 1.4 mg/mL

Best for

Quick measurements in simple buffers without much detergent.

Common interference

Sensitive to detergents such as SDS; weaker response across very different protein compositions.

Key question

Why does the curve bend once dye-binding sites become limiting?

Standard curve playground

Use the preset reference curve to explore interpolation, dilution factor, and why extrapolation is risky.

Concentration (mg/mL)	Absorbance
0	0.041
0.125	0.117
0.25	0.208
0.5	0.394
0.75	0.573
1	0.742

Unknown absorbanceSample dilution factor

Interpolated concentration0.598 mg/mL

Original sample concentration2.991 mg/mL

Within the recommended range.

Approximate reference standard curve

Protein concentration (mg/mL) · Absorbance

Points to check

Different assays are not interchangeable: chemistry determines what interferes and what concentration range behaves linearly.
A strong R² does not make extrapolation safe. If the unknown lies outside the standard range, dilution strategy matters more than algebra.
Reported concentration should follow the workflow: read absorbance → interpolate from the curve → multiply back by the dilution factor.