The Lowry Assay - Commonly used protein assay methods

References: 

O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) J. Biol. Chem. 193: 265. (The original method)

Hartree E. E. (1972).  Anal. Biochem.  48:  422 (This modification makes the assay linear over a larger range than the original assay)

J.R. Dulley and P.A. Grieve (1975) Anal. Biochem. 64: 136. (This is a useful modification of the original Lowry method that includes 0.5% sodium dodecylsulfate in the alkaline reagent.  This obviates interference from many detergents and helps disperse membranes in the sample.)

A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70: 241.  (Another useful modification of the original Lowry method that can be useful when the solution contains interfering contaminants.  The proteins in the samples are precipitated by a mixture of sodium deoxycholate and trichloroacetic acid and centrifugation prior to assay.  If the contaminants stay in the supernatant they can be removed and the amount of precipitated protein determined.

Quick Guide

How does it work?

• The first step is a Biuret reaction which reduces Cu⁺² to Cu⁺¹
• The second reaction uses Cu⁺¹ to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate).  This is detectable in the range of 500 to 750 nm

Detection Limitations

• 2-100 µg

Advantages

• Sensitive over a wide range
• The most commonly referenced procedure for protein determination
• Can be performed at room temperature
• 10-20 times more sensitive than UV detection
• Can be performed in a microplate format

Disadvantages

• Many substances interfere with the assay
• Alkaline copper reagent is laborious to prepare and will develop carbonate scales over storage which interfere with optical activity, thus it must be prepared fresh daily
• Takes a considerable amount of time to perform
• The assay is photosensitive, so illumination during the assay must be kept consistent for all samples
• Amount of color varies with different proteins

General Considerations

• Some researchers have reported that repeated assays in the same cuvettes cause them to be etched
• Many chemical distributors sell a modified Lowry assay that is more stable and sensitive than homemade versions
• Since reduced copper is detected in the procedure, make sure that the distilled water used in the procedure is fed from plastic lines and not copper lines.  In general water from 18 megaohm water polishers is satisfactory
• Variation in the content of tyrosine and tryptophan residues will influence the assay

Procedure

Alkaline Reagent

0.1 M NaOH

2% Na₂CO₃

0.5% Na Tartrate   (use of potassium salt will cause SDS to be insoluble)

0.5% Na Dodecylsulfate

Copper Reagent

1% CuSO₄*5H₂O

Assay Mix (MAKE FRESH EACH DAY)

50 mL alkaline reagent and 0.5 mL copper reagent

Folin-Ciocalteu Reagent

Dilute with an equal volume of water to prepare the desired volume

Procedure:

• Add samples containing up to 100 µg of protein
• Bring all tubes to 1 mL total volume with water.  Be sure to have two tubes containing only water as blanks.  Also use reagent or buffer blanks if needed.
• Prepare the Assay Mix and diluted Folin-Ciocalteu reagent.
• To each tube add 5 mL of assay mix and thoroughly vortex.
• Incubate tubes at room temperature for 10 min.
• Add 0.5 mL of diluted Folin-Ciocalteu reagent.  Vortex immediately.
• Incubate at room temperature for 30 min.
• Vortex the tubes, zero the spectrophotometer with the blank and measure absorbance at 660 nm (or other appropriate wavelength).  The data from the standard curve are usually linear enough that a straight-line interpolation can be used to determine the concentration of unknowns.

Discussion

The Lowry method relies on two different reactions.  The first is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions.  This is called a "Biuret" chromophore.  The second is the reduction of Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and tryptophan residues.  The reduced Folin-Ciocalteu reagent is blue and thus detectable with a spectrophotometer in the range of 500-750 nm.  The Biuret reaction itself is not all that sensitive.  Using the Folin-Ciocalteu reagent to detect reduced copper makes the assay nearly 100 times more sensitive than the Biuret reaction alone. 

The assay is relatively sensitive, but takes more time than other assays and is susceptible to many interfering compounds.  The following substances are known to interfere with the Lowry assay: detergents, carbohydrates, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl compounds, disulfide compounds, magnesium and calcium.  Most of these interfering substances are commonly used in buffers for preparing proteins.  This is one of the major limitations of the assay.  The Lowry assay is sensitive to variations in the content of tyrosine and tryptophan residues.  If the protein you are assaying has an unusual content of these residues, an appropriate substitute standard is required.   The standard curve is linear in the 1 to 100 ug protein region.  The absorbance can be read in the region of 500 to 750 nm.  Most researchers use 660 nm, but other wavelengths also work and may reduce the effects of contamination (e.g. chlorophyll in plant samples interferes at 660 nm, but not at 750 nm).

Typical Standard Curve for a Lowry Assay