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Lowry protein assay using an automatic microtitre plate assay

Article  in  Analytical Biochemistry · April 1986

DOI: 10.1016/0003-2697(86)90090-4 · Source: PubMed

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ANALYTICAL BIOCHEMISTRY 153,262-266 (1986)

Lowry Protein Assay Using an Automatic Microtiter

Plate Spectrophotometer

HUGHJ.L.FRYER,‘GEORGE E. DAVIS,MARSTON MANTHORPE,*ANDSILVIOVARON

Department of Biology, School of Medicine, University of California at San Diego, Lu Jolla. California 92093
Received August 20, 1985

The method of protein determination reported by Lowry et al. (195 1, J. Biol. Chem. 193,265
275) has been adapted for use with 96-well microtiter plates and an automatic microplate spec-
trophotometer. The spectrophotometer has been interfaced with a computer which plots the
standard curve and calculates the protein content of each sample. The adapted method offers
advantages over previously reported methods in that it is more rapid and uses a smaller sample
volume (100 ~1) for samples containing 3-300 &ml (0.3-30 pplassay) of protein. The method
of Bensadoun and Weinstein (1976, Anal. Biochem. 70, 241-252) for precipitating microgram
amounts of protein away from substances which interfere with the Lowry assay has also been
adapted to this microplate procedure. These techniques should be particularly useful for laboratories
where large numbers of samples containing a wide range of protein concentrations are assayed.
8 1986 Academic F’ra, Inc.
KEY WORDS: protein determination; automatic spectrophotometer; protein titration.

The method of Lowry et al. (1) is a widely
used procedure for the determination of pro-
tein concentration. Although several sub-
stances are reported to interfere with the
Lowry method [for review see (2)], Bensadoun
and Weinstein (3) have reported a modifica-
tion of the method whereby microgram
amounts of protein are quantitatively precip-
itated away from most interfering substances.
One inconvenience of both of these methods,
particularly when large numbers of samples
are to be assayed, is the relatively long period
of time required to determine sample absor-
bances and calculate pr&ein content.
In this study, we describe adaptations of the
above protein assay methods which permit
rapid absorbance determination and computer
data analysis of a large number of samples.
The procedures rely on the use of 96-well mi-
crotiter plates and an automatic microplate
’ Present address: Biological Sciences Group, Depart-
ment of Physiology, University of Connecticut, Storm,
Conn. 06268.
’ To whom correspondence should be addressed.
spectrophotometer interfaced with a com-
puter.
MATERIALS AND METHODS
Reagents.The copper tartrate solution (I),
which was concentrated five times, was made
fresh daily by mixing previously prepared
stock solutions of, respectively, 5% CuSO4,
10% Na,K-tartrate, and 10% Na2C04 in 0.5
N NaOH ( 1: 1: 100). The Folin-Ciocalteu phe-
nol reagent (Sigma) was diluted immediately
before use with 0.1 N NaOH. Sodium deoxy-
cholate (DOC)3 was purchased from Sigma
and trichloroacetic acid (TCA) from Mal-
linckrodt. Bovine serum albumin (BSA, frac-
tion V, Calbiochem) was mixed with water and
filter sterilized through a 0.22-pm Millex filter,
and its concentration was appropriately ad-
justed using an A2s0 of 0.66 for 1 mg/ml of
3 Abbreviations used: DOC, deoxycholate; TCA, tri-
chloroacetic acid, BSA, bovine serum albumin; A~W, op
tical density at 630 nm; SDS, sodium dodecyl sulfate;
Hepes, l -(2-hydroxyethyl)- 1-piperazineethanesulfonic
acid.

0003-2697186$3.00 262
CopyrightQ 1986 by Academic F’rcsq Inc.
All rights of reproduction in any form xwwd.
 AUTOMATED
BSA (4). Aliquots of the BSA solution were
stored at -20°C before use.
Normal microplate Lowry protein assay.
Reaction vessels, which also served as cuvettes,
were the individual 4.5-mm-diameter wells of
96-well microtiter plates (A/2 Costar No.
3690). Protein solutions were placed into each
well to a final volume of 100 ~1, mixed with
25 ~1 of the 5X copper tartrate solution, and
the mixture was incubated for 10 min at room
temperature. A lo-p1 amount of diluted phe-
nol reagent (see above) was then added and
the stirred mixture incubated for 20 min at
room temperature. Serial dilutions of the
standard BSA solution were prepared in suc-
cessive wells and treated similarly. All addi-
tions, mixings, and dilutions were performed
using a 12-channel micropipet (Titer Tech).
Optical density at 630 nm was determined us-
ing a Dynatech MR 600 microplate reader set
without a reference wavelength at a 1.00 cal-
ibration and a 1.99 threshold. Data from the
spectrophotometer were fed directly to an Ap-
ple IIe computer and analyzed using the Im-
munosoft program (Dynatech). The standard
curve was constructed as described by Stauffer
(5) as the log of the absorbance at 630 nm
(A& versus the log of the total protein in mi-
crograms per well and the protein content
in micrograms of each well was calculated and
printed out.
Modjied microplate Lowry protein assayfor
the removal of interfering substances. In those
cases where samples contain substances
known or suspected to interfere with the
Lowry protein assay, the following procedure
can be used to remove the interference. This
method is an adaptation of the procedure de-
scribed by Bensadoun and Weinstein (3). Pro-
tein samples were added, with or without di-
lution, to a final volume of 100 ~1 to the wells
of a V-bottom microtiter plate (Costar). A lo-
~1 amount of 0.35% DOC was mixed with the
samples and the mixtures were incubated for
10 min at room temperature. The sample pro-
teins were precipitated by the addition of 30
~1 of 30% TCA and incubated for another 10
min. The plates were then sealed with adhe-
PROTEIN ASSAY 263
sive-backed microtiter plate covers (Costar)
and centrifuged at 900g for 30 min in a mi-
croplate holder. The supematants were re-
moved and 100 ~1 of water was added to the
precipitates. The above precipitation was car-
ried out a second time, 100 ~1 of water was
then added to the sample, and the above de-
scribed Lowry protein assay was carried out.
The V-bottom microplates were used instead
of flat-bottom plates because the conical well
accumulates a tighter precipitate which re-
mains unperturbed during supematant re-
moval. After completion of the color reaction,
the samples were transferred to A/2 microtiter
plates and read on the spectrophotometer, and
the data were analyzed as described above.
Sample volume can be increased to 150 ~1 if
the volume of both DOC and TCA similarly
are increased by 50%.
RESULTS
Linearity of the normal microplate Lowry
protein assay. A linear-scale plot of the absor-
bance at 630 nm versus BSA concentration
over the range of 0.3-30 /Ig protein/well (3-
300 pg protein/ml in the original sample) is
shown in Fig. 1A. Although the resulting stan-
dard curve may appear linear over discrete
protein concentration ranges, the overall curve
is nonlinear. When the same data are plotted
using double-log coordinates, the curve is lin-
ear over the entire measured range, in agree-
ment with other reports (5,6). The standard
curves obtained using ovalbumin, cytochrome
c, or a mixture of BSA, ovalbumin, and cy-
tochrome c, or even fetal calf serum were
identical to those of BSA alone (data not
shown). The log-log plot was adopted in the
present study because it was more amenable
to the computer data analysis.
Modified microplate protein determination
after removal of interfering substances. Certain
substances, such as Hepes buffer [ l-(2-hy-
droxyethyl)- 1-piperazineethanesulfonic acid]
have been reported to interfere with the Lowry
protein assay (2) and the Bensadoun and
Weinstein (3) modification allows for proteins
264 FRYER ET AL.

1.E

1.2

::
k”
0.8

0.4

10 20 30 40 1 10 100
ug Protein/O.1 ml ug Protein/ 0.1 ml

FIG. 1. Standard curves for the determination of BSA protein concentration using the normal microplate
Lowry assay.The same data are presented using linear (A) or log-log (B) coordinates. Absorbance values
are averages from duplicate assaysof four separately prepared protein solutions assayed on different days.
Error bars represent standard deviations from all eight values.

to be precipitated away from most interfering assay (Table 1, compare A and B). The inter-
substances. We have adapted this precipitation fering substances could be categorized into two
procedure (3) to the microplate assay (see Ma- groups depending on whether they increased
terials and Methods). Figure 2 shows the stan- (Table 1C) or decreased (Table 1D) the ab-
dard log-log plot for BSA derived from assays sorbance observed before precipitation. In
performed in the presence (triangles) or ab- both cases, however, the precipitation tech-
sence (circles) of 0.3 M Hepes, either before nique removed the interference and allowed
(open symbols) or after (closed symbols) the
two precipitation steps. In the presence of
Hepes but without precipitation (open trian-
gles), the absorbance values of the blanks and
. i
of all the protein concentrations tested ex- 1.0: 6
ceeded 2.00, the maximum readable value on b”
the spectrophotometer. After precipitation,
however, the absorbance at each protein con-
centration was restored to that obtained in the
absenceof Hepes (Fig. 2, compare open and
closed circles with closed triangles). The vari-
ability of replicable absorbance readings for
different protein concentrations was similar to 0.01
0.1 1 10 100
that shown in Fig. 1B.
The precipitation technique was also used pg Protein/O.1 ml

to assay BSA protein in samples containing
other potentially interfering substances besides
Hepes. Table 1 shows a comparison of the ab-
sorbance values obtained when 6 pg of BSA
was assayed either before or after precipitation.
Two substances, 3 M urea and 1% sodium do-
decyl sulfate (SDS), did not affect the normal
FIG. 2. Standard curves for the determination of protein
concentration in the presence or after removal of an in-
terfering substance. Assays of the indicated BSA concen-
trations were performed in the absence (circles) or presence
(triangles) of 0.3 M Hepes and either before (open symbols)
or after (closed symbols) two successive TCA-DOC pre-
cipitations (see Materials and Methods). Values are back-
ground-corrected averages of triplicate determinations.
 AUTOMATED PROTEIN ASSAY 265

TABLE I
MICROPLATE LOWRY PROTEINASSAYSINTHEPRESENCE
OFPOTENTIAL INTERFERINGSUBSTANCES

Standard Precipitation
aw assaya

Additive

0.46 1 100 0.482 100

.<:I /
A. No additive LOG (OD) Versus LOG (Concentration)

B. 3 M Urea 0.492 107 - -

1% SDS 0.426 92 - -
C. 0.3 M Hepes >2.0d >433 0.452 94
0.15% Phenol >2.0d >433 0.488 101
1.O% Ampholine 22.0d >433 0.463 96
Culture medium’ >2.0d >433 0.491 102
0.3 M Tris-HCI >2.0d >433 0.438 91
concentration
D. 45% Sucrose 0.156 34 0.440 91
C
15% (NH&SO4 0.09 1 20 0.460 95
Tl over T5 over 19 over Tl32800 T17under
’ Using the Bensadoun and Weinstein (3) modification TZ over T6 over TlO5110 T 14 0.280 T I.3 under
T3 over T7 6311 ill 0498 515 under Tl9 under
of the Lowry protein assay after removal of interfering
T4 7903 TR 0624 TIZOOSZ Tl6 under T20 under
substances (see Materials and Methods).
* The indicated absorbance values are background-cor- mg/ml protein
rected averages of triplicate protein assaysof 6 rg of BSA. 1 11 ,I, iv "
’ Values are % of&, relative to the control values with- 790 6.28 a510 0.028 < 0003
out potential interfering substance.
d AsM Values exceeded the maximal readable absorbance FIG. 3. One suggested format for arranging standard
of the spectrophotometer. and test samples in 96-well microplates and computer
e Dulbecco’s modified Eagle’s basal medium. output of calculated protein values using the automatic
spectrophotometer method. (A) Duplicate serial dilutions
of fetal calf serum solutions are provided sequentially in
wells A l-2 through G l-2 (hatched wells) while wells H l-
the accurate assay of protein concentration 2 (stippled wells) are not provided with protein. Five sam-
over the range of the entire standard curve ples (i-v) were titrated serially IO-fold in duplicate as fol-
(data not shown). lows: All wells in columns 3- 12 receive 100 ~1 water. The
Computer analysis of the data. Figure 3 il- water in row A is replaced with 111.1 ~1 of test sample,
lustrates one particular format for. arranging mixed, and 11.1 pl is transferred to row B, mixed again,
and the transfers continued through row D until the last
the standard and test samples for analysis using 11.1 *cl discarded. This arrangement will provide sample
the Dynatech Immunosoft program. Test dilutions covering a IOOO-fold range and ensure that at
samples (i-v) are presented to the wells in four least one sample protein concentration will fall within the
successive, duplicate 1O-fold dilutions (Fig. 3A, range covered by the standard curve. (B) Computer output
wells A-D, labeled Tl-4, T5-8, T9-12, T13- of the log-log standard curve for BSA covering the range
16, and T17-20) to assure that at least one 0.3-30 &well. (C) Computer output format for average
protein concentration of each duplicate setof dilutons (Tl-
absorbance value falls within the log-log stan- 20) in &O. 1 ml/well. The concentration of protein in rcg/
dard curve for BSA (Fig. 3B) and thus is con- ml of each original sample (i-v) or its 10 to IOffold di-
vertible by the program to micrograms of pro- lution is not printed out by the computer but is a multi-
tein per well (Fig. 3C). The format has the plicand of IO and is shown for each sample below the
advantage that virtually any test sample having output data. The readings of “over” and “under” are given
when the sample absorbances fall outside of the standard
a protein concentration above 0.003 mg/ml curve (i.e., 1.5 or 0.003, respectively). For example, sample
can be assayed without having to carry out v had too low a protein concentration (i.e., co.003 mg/
preliminary determinations to estimate the ml) to be detected.
266 FRYER ET AL.
gross sample protein concentration range. Be-
cause all dilutions and sample volumes are di-
visible by 10, it is a simple matter to inspect
the values and convert them to milligrams per
milliliter protein in the original sample. The
total elapsed time required to read each 96-
well plate and calculate, print, and estimate
the corresponding milligrams per milliliter
protein values is less than 5 min.
DISCUSSION
In the microtiter plate Lowry protein assay
described here, there are three steps which sig-
nificantly shorten the assay time, namely (i)
the addition and mixing of reagents by the use
of a multichannel pipet, (ii) the absorbance
determination of the samples by the use of a
microtiter plate spectrophotometer, and (iii)
the analysis of the data with a computer in-
terfaced with the spectrophotometer. Samples
and reagents are added and mixed in micro-
wells, which also serve as cuvettes, thus elim-
inating time-consuming transfers. Since the
total number of samples to be assayed is not
a particular inconvenience, test samples con-
taming very high and/or unpredictable protein
concentrations can be serially diluted in suc-
View publication stats
cessive wells to ensure that at least one absor-
bance value falls within the range of the stan-
dard curve, thus conserving sample and elim-
inating the necessity of trial protein range
determinations. Computer analysis and the
printout of protein values is also more rapid
and less subject to calculation errors. The mi-
croplate Lowry protein determination method
should be of particular value for laboratories
where large numbers of samples are processed,
and where a microplate spectrophotometer is
already in use for other purposes.
ACKNOWLEDGMENTS
This work was supportedby NINCDS Grant NS 16349,
and NSF Grants BNS 82- 18366 and 85-02 158.
REFERENCES
1. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and
Randall, R. J. (195 I) J. Biol. Chem. 193,265-275.
2. Peterson. G. L. ( 1979) Anal. Biochem. 100.20 l-220.
3. Bensadoun, A.; and Weinstein, D. (1976) Anal.
Biochem. 70,241-252.
4. Sober, H. A. (1970) Handbook of Biochemistry, 2nd
ed., Chemical Rubber Co., Cleveland, Ohio.
5. Stauffer, C. F. (1975) Anal. Biochem. 69, 646-648.
6. Bates, W. K., and McAllister, D. F. (1974) Anal.
Biochem. 30,386-390.