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The Lowry method for protein quantitation.

Article  in  Methods in Molecular Biology · January 1984

DOI: 10.1385/0-89603-062-8:1 · Source: PubMed

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Preface
The Protein Protocols Handbook, Second Edition aims to provide a cross-section of
analytical techniques commonly used for proteins and peptides, thus providing a
benchtop manual and guide for those who are new to the protein chemistry laboratory
and for those more established workers who wish to use a technique for the first time.
All chapters are written in the same format as that used in the Methods in Molecular
Biology™ series. Each chapter opens with a description of the basic theory behind the
method being described. The Materials section lists all the chemicals, reagents, buffers,
and other materials necessary for carrying out the protocol. Since the principal goal of
the book is to provide experimentalists with a full account of the practical steps necessary
for carrying out each protocol successfully, the Methods section contains detailed step-
by-step descriptions of every protocol that should result in the successful execution of
each method. The Notes section complements the Methods material by indicating how
best to deal with any problem or difficulty that may arise when using a given technique,
and how to go about making the widest variety of modifications or alterations to the
protocol.
Since the first edition of this book was published in 1996 there have, of course, been
significant developments in the field of protein chemistry. Hence, for this second edition
I have introduced 60 chapters/protocols not present in the first edition, significantly
updated a number of chapters remaining from the first edition, and increased the overall
length of the book from 144 to 164 chapters. The new chapters particularly reflect the
considerable developments in the use of mass spectrometry in protein characterization.
Recognition of the now well-established central role of 2-D PAGE in proteomics has
resulted in an expansion of chapters on this subject, and I have also included a number
of new techniques for staining and analyzing protein blots. The section on glycoprotein
analysis has been significantly expanded, and aspects of single chain antibodies and
phage-displayed antibodies have been introduced in the section on antibodies.
We each, of course, have our own favorite, commonly used methods, be it a gel
system, gel-staining method, blotting method, and so on; I’m sure you will find yours
here. However, I have, as before, also described alternatives for some of these tech-
niques; though they may not be superior to the methods you commonly use, they may
nevertheless be more appropriate in a particular situation. Only by knowing the range of
techniques that are available to you, and the strengths and limitations of these techniques,
will you be able to choose the method that best suits your purpose. Good luck in your
protein analysis!
John M. Walker

v
 The Lowry Method 7

2
The Lowry Method for Protein Quantitation

Jakob H. Waterborg

1. Introduction
The most accurate method of determining protein concentration is probably acid
hydrolysis followed by amino acid analysis. Most other methods are sensitive to the
amino acid composition of the protein, and absolute concentrations cannot be obtained
(1). The procedure of Lowry et al. (2) is no exception, but its sensitivity is moderately
constant from protein to protein, and it has been so widely used that Lowry protein
estimations are a completely acceptable alternative to a rigorous absolute determina-
tion in almost all circumstances in which protein mixtures or crude extracts are
involved.
The method is based on both the Biuret reaction, in which the peptide bonds of
proteins react with copper under alkaline conditions to produce Cu+, which reacts with
the Folin reagent, and the Folin–Ciocalteau reaction, which is poorly understood but in
essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the
copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong
blue color, which depends partly on the tyrosine and tryptophan content. The method is
sensitive down to about 0.01 mg of protein/mL, and is best used on solutions with
concentrations in the range 0.01–1.0 mg/mL of protein.

2. Materials
1. Complex-forming reagent: Prepare immediately before use by mixing the following stock
solutions in the proportion 100:1:1 (by vol), respectively:
Solution A: 2% (w/v) Na2CO3 in distilled water.
Solution B: 1% (w/v) CuSO4·5H2O in distilled water.
Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. 2 N NaOH.
3. Folin reagent (commercially available): Use at 1 N concentration.
4. Standards: Use a stock solution of standard protein (e.g., bovine serum albumin fraction V)
containing 2 mg/mL protein in distilled water, stored frozen at –20°C. Prepare standards
by diluting the stock solution with distilled water as follows:
Stock solution (µL) 0 2.5 5 12.5 25 50 125 250 500
Water (µL) 500 498 495 488 475 450 375 250 0
Protein conc. (µg/mL) 0 10 20 50 100 200 500 1000 2000

From: The Protein Protocols Handbook, 2nd Edition

Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ

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 8 Waterborg

3. Method
1. To 0.1 mL of sample or standard (see Notes 1–4), add 0.1 mL of 2 N NaOH. Hydrolyze at
100°C for 10 min in a heating block or boiling water bath.
2. Cool the hydrolysate to room temperature and add 1 mL of freshly mixed complex-form-
ing reagent. Let the solution stand at room temperature for 10 min (see Notes 5 and 6).
3. Add 0.1 mL of Folin reagent, using a vortex mixer, and let the mixture stand at room
temperature for 30–60 min (do not exceed 60 min) (see Note 7).
4. Read the absorbance at 750 nm if the protein concentration was below 500 µg/mL or at
550 nm if the protein concentration was between 100 and 2000 µg/mL.
5. Plot a standard curve of absorbance as a function of initial protein concentration and use it
to determine the unknown protein concentrations (see Notes 8–13).

4. Notes
1. If the sample is available as a precipitate, then dissolve the precipitate in 2 N NaOH and
hydrolyze as described in Subheading 3, step 1. Carry 0.2-mL aliquots of the hydrolyzate
forward to Subheading 3, step 2.
2. Whole cells or other complex samples may need pretreatment, as described for the Burton
assay for DNA (3). For example, the perchloroacetic acid (PCA)/ethanol precipitate from
extraction I may be used directly for the Lowry assay, or the pellets remaining after the
PCA hydrolysis step (Subheading 3, step 3 of the Burton assay) may be used for Lowry.
In this latter case, both DNA and protein concentration may be obtained from the same
sample.
3. Peterson (4) has described a precipitation step that allows the separation of the protein
sample from interfering substances and also consequently concentrates the protein sample,
allowing the determination of proteins in dilute solution. Peterson’s precipitation step is
as follows:
a. Add 0.1 mL of 0.15% deoxycholate to 1.0 mL of protein sample.
b. Vortex-mix, and stand at room temperature for 10 min.
c. Add 0.1 mL of 72% trichloroacetic acid (TCA), vortex-mix, and centrifuge at 1000–
3000g for 30 min.
d. Decant the supernatant and treat the pellet as described in Note 1.
4. Detergents such as sodium dodecyl sulfate (SDS) are often present in protein prepara-
tions, added to solubilize membranes or remove interfering substances (5–7). Protein pre-
cipitation by TCA may require phosphotungstic acid (PTA) (6) for complete protein
recovery:
a. Add 0.2 mL of 30% (w/v) TCA and 6% (w/v) PTA to 1.0 mL of protein sample.
b. Vortex-mix, and stand at room temperature for 20 min.
c. Centrifuge at 2000g and 4°C for 30 min.
d. Decant the supernatant completely and treat the pellet as described in Note 1.
5. The reaction is very pH dependent, and it is therefore important to maintain the pH between
10 and 10.5. Therefore, take care when analyzing samples that are in strong buffer outside
this range.
6. The incubation period is not critical and can vary from 10 min to several hours without
affecting the final absorbance.
7. The vortex-mixing step is critical for obtaining reproducible results. The Folin reagent is
reactive only for a short time under these alkaline conditions, being unstable in alkali, and
great care should therefore be taken to ensure thorough mixing.
8. The assay is not linear at higher concentrations. Ensure that you are analyzing your sample
on the linear portion of the calibration curve.

02/Waterborg/7-9F[12.20.1] 8 12/27/2001, 12:01 PM

 The Lowry Method 9

9. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate
or triplicate unknowns are recommended.
10. One disadvantage of the Lowry method is the fact that a range of substances interferes
with this assay, including buffers, drugs, nucleic acids, and sugars. (The effect of some of
these agents is shown in Table 1 in Chapter 3.) In many cases, the effects of these agents
can be minimized by diluting them out, assuming that the protein concentration is suffi-
ciently high to still be detected after dilution. When interfering compounds are involved,
it is, of course, important to run an appropriate blank. Interference caused by detergents,
sucrose, and EDTA can be eliminated by the addition of SDS (5) and a precipitation step
(see Note 4).
11. Modifications to this basic assay have been reported that increase the sensitivity of the
reaction. If the Folin reagent is added in two portions, vortex-mixing between each addi-
tion, a 20% increase in sensitivity is achieved (8). The addition of dithiothreitol 3 min
after the addition of the Folin reagent increases the sensitivity by 50% (9).
12. The amount of color produced in this assay by any given protein (or mixture of proteins)
is dependent on the amino acid composition of the protein(s) (see Introduction). There-
fore, two different proteins, each for example at concentrations of 1 mg/mL, can give
different color yields in this assay. It must be appreciated, therefore, that using bovine
serum albumin (BSA) (or any other protein for that matter) as a standard gives only an
approximate measure of the protein concentration. The only time when this method gives
an absolute value for protein concentration is when the protein being analyzed is also used
to construct the standard curve. The most accurate way to determine the concentration of
any protein solution is amino acid analysis.
13. A means of speeding up this assay using raised temperatures (10) or a microwave oven
(see Chapter 5) has been described.

References
1. Sapan, C. V., Lundblad, R. L., and Price, N. C. (1999) Colorimetric protein assay tech-
niques. Biotechnol. Appl. Biochem. 29, 99–108.
2. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measure-
ment with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
3. Waterborg, J. H. and Matthews, H. R. (1984) The Burton assay for DNA, in Methods in
Molecular Biology, Vol. 2: Nucleic Acids (Walker, J. M., ed.), Humana Press, Totowa, NJ,
pp. 1–3.
4. Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95–121.
5. Markwell, M.A.K., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Protein determi-
nation in membrane and lipoprotein samples. Methods Enzymol. 72, 296–303.
6. Yeang, H. Y., Yusof, F., and Abdullah, L. (1998) Protein purification for the Lowry assay:
acid precipitation of proteins in the presence of sodium dodecyl sulfate and other biologi-
cal detergents. Analyt. Biochem. 265, 381–384.
7. Chang, Y. C. (1992) Efficient precipitation and accurate quantitation of detergent-solubi-
lized membrane proteins. Analyt. Biochem. 205, 22–26.
8. Hess, H. H., Lees, M. B., and Derr, J. E. (1978) A linear Lowry-Folin assay for both water-
soluble and sodium dodecyl sulfate-solubilized proteins. Analyt. Biochem. 85, 295–300.
9. Larson, E., Howlett, B., and Jagendorf, A. (1986) Artificial reductant enhancement of the
Lowry method for protein determination. Analyt. Biochem. 155, 243–248.
10. Shakir, F. K., Audilet, D., Drake, A. J., and Shakir, K. M. (1994) A rapid protein determi-
nation by modification of the Lowry procedure. Analyt. Biochem. 216, 232–233.

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