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Short answer
The Lowry assay is a colorimetric method for measuring protein concentration. Protein reacts with copper under alkaline conditions, and the reduced copper then reacts with Folin reagent to give a blue color read in a spectrophotometer. It works best between 0.01 and 1.0 mg/mL of protein, and many common buffer components interfere with it.
Definition and scope
The Lowry method, from the 1951 Folin phenol reagent paper by Lowry, Rosebrough, Farr and Randall (J. Biol. Chem. 193, 265-275), is a protein quantitation assay. The Hebrew University Protein Purification Facility describes it as based on two reactions together: the biuret reaction, in which peptide bonds react with copper under alkaline conditions to produce Cu+, and the Folin-Ciocalteu reaction, in which phosphomolybdotungstate is reduced to a blue product by copper-catalyzed oxidation of aromatic amino acids. The color therefore depends partly on tyrosine and tryptophan content.
Thermo Fisher’s overview of protein assays classes Lowry as a biuret-type (copper-based) method: protein-copper chelation followed by secondary detection of reduced copper. That puts it in a different chemical family from dye-binding methods such as the Bradford assay.
How the Lowry assay is used in practice
The Hebrew University protocol, adapted from Protein Protocols on CD-ROM (Humana Press, 1998), gives the working parameters below. This is a summary of the published protocol for orientation, not a bench procedure.
- Complex-forming reagent: three stock solutions (2% Na2CO3, 1% CuSO4·5H2O and 2% sodium potassium tartrate) mixed 100:1:1 and prepared immediately before use. A 2N NaOH solution and a commercially available Folin reagent, diluted 1:2 in water, are also needed.
- Standard: bovine serum albumin (fraction V) at a 4 mg/mL stock, diluted into a series from 0 to 2000 µg/mL.
- Timing: sample or standard is mixed with the complex-forming reagent for 10 minutes at room temperature; diluted Folin reagent is added with vortexing and the mixture stands 30-60 minutes, not exceeding 60.
- Reading: absorbance at 750 nm for protein below 500 µg/mL, or 550 nm for 100-2000 µg/mL, read against a standard curve of absorbance versus protein concentration.
- Range: sensitive to about 0.01 mg/mL and best used between 0.01 and 1.0 mg/mL.
The protocol also notes that mixing the Folin reagent by vortexing is critical because the reagent is unstable in alkali, and that pH must stay between 10 and 10.5. For building and fitting the curve itself, see the site’s guides on the Bradford standard curve and the BCA standard curve, which use the same absorbance-versus-concentration logic. A general primer on the instrument is in UV-Vis spectrophotometer basics.
Lowry vs Bradford and related assays
Lowry vs Bradford assay
The two assays differ in chemistry, and that determines what each tolerates. Per Thermo Fisher’s overview, the Bradford assay needs no copper-protein binding, is fast, runs at room temperature, is compatible with reducing agents and is incompatible with surfactants, with higher protein-to-protein variation than copper-based assays. The Lowry assay, being copper-based, is incompatible with substances that reduce copper, including DTT. On detergents the two sources disagree for Lowry: the Hebrew University protocol lists many detergents among its interferents, whereas Thermo Fisher’s overview lists compatibility with most surfactants, so check the detergent type and concentration in the protocol you follow.
| Feature | Lowry | Bradford | Source |
|---|---|---|---|
| Chemistry | Biuret-type: protein-copper chelation, then secondary detection of reduced copper | Dye binding, no copper-protein binding needed | Thermo Fisher overview |
| Reducing agents (e.g. DTT) | Incompatible | Compatible | Thermo Fisher overview |
| Surfactants | Sources differ: the Hebrew University protocol lists many detergents as interferents, while Thermo Fisher lists compatibility with most surfactants | Incompatible | Hebrew University protocol; Thermo Fisher overview |
| Protein-to-protein variation | Lower than Coomassie dye-based assays | Higher than copper-based assays | Thermo Fisher overview |
Bradford vs Lowry assay: which fits a sample
The table gives the practical split. A sample containing DTT or another reducing agent points away from Lowry; a sample containing detergent points away from Bradford, and for Lowry the answer depends on which source and detergent you rely on, with the protocol’s remedies listed below. The same Thermo Fisher page shows that copper-based assays share similar protein-to-protein variation. The site’s micro BCA assay guide covers a related copper-based format.
Interferences and the remedies the protocol lists
The Hebrew University protocol states that a range of substances interfere with Lowry, including buffers, drugs, nucleic acids and sugars, and lists specific ones: many detergents, urea, guanidine HCl, high sucrose, ammonium sulphate, Tris-HCl above 0.1 M, sodium acetate or sodium phosphate above 1 M, EDTA and reducing agents. It gives the following remedies.
| Problem | Remedy stated in the protocol |
|---|---|
| Interfering compound in general | Dilute it out, if protein stays detectable after dilution; run an appropriate blank |
| Detergents, sucrose, EDTA | Add SDS; the protocol’s preferred alternative is Lowry-TCA or Peterson precipitation |
| Interferents plus dilute protein | Peterson precipitation (deoxycholate, then TCA, then centrifugation, then dissolving the pellet in 2N NaOH), which separates protein from interferents and concentrates it |
The same protocol reports two sensitivity modifications: adding the Folin reagent in two portions raises sensitivity by about 20%, and adding dithiothreitol 3 minutes after the Folin reagent raises it by about 50%.
Where it matters for labs
The reagent list is the practical point for lab purchasing and inventory: the assay needs a commercially available Folin reagent, the three copper-carbonate-tartrate stocks, sodium hydroxide, a protein standard such as BSA fraction V, and a spectrophotometer able to read at 750 nm and 550 nm. Which wavelength is read depends on the expected concentration, so the expected range of your samples determines the setup. Wavelength accuracy of the reader is a separate quality question, covered in spectrophotometer calibration.
Common misunderstandings
- “The result is the absolute protein concentration.” The color produced depends on amino acid composition, so two proteins at the same concentration can give different color yields. A BSA standard gives only an approximate concentration for other proteins; the protocol says the value is absolute only when the protein being measured is also used to build the standard curve, and that amino acid analysis is the most accurate way to determine concentration.
- “Longer incubation is always safe.” The protocol’s stated timing is 30-60 minutes after Folin addition, not to exceed 60.
- “It works at any concentration.” The protocol states the response is not linear at higher concentrations, which is why a 0.01-1.0 mg/mL working range is given.
- “Lowry and Bradford are interchangeable.” Their interferents differ, as shown above.
FAQ
What is the Lowry assay used for?
Measuring the protein concentration of a solution by color development against a standard curve.
What wavelength is the Lowry assay read at?
The Hebrew University protocol reads at 750 nm for protein below 500 µg/mL and at 550 nm for 100-2000 µg/mL.
Lowry vs Bradford assay: which tolerates reducing agents?
Bradford. Thermo Fisher lists Bradford as compatible with reducing agents and Lowry as incompatible with substances that reduce copper, including DTT.
Which substances interfere with the Lowry assay?
Many detergents, urea, guanidine HCl, high sucrose, ammonium sulphate, Tris-HCl above 0.1 M, high concentrations of sodium acetate or phosphate, EDTA and reducing agents, among others.
Sources
- Hebrew University of Jerusalem, Wolfson Centre for Applied Structural Biology, Protein Purification Facility, “Lowry – Protein Determination”: wolfson.huji.ac.il/purification/Protocols/loury.html (checked 2026-10-29). The page cites Waterborg and Matthews, Protein Protocols on CD-ROM, Humana Press, 1998, and Lowry et al., J. Biol. Chem. 193, 265-275 (1951).
- Thermo Fisher Scientific, “Overview of protein assays” (Pierce protein methods): thermofisher.com, Overview of protein assays (checked 2026-10-29).
