ANALYTICAL BIOCHEMISTRY 31, 146-148 (1969)

A Simple Calorimetric Method for Determination

of Protein’
S. BRAMHALL, N. NOACK, M. WU, AND J. R. LOEWENBERG
Department of Botany, University of Wisconsin-Milwaukee,
Milwaukee, Wisconsin 53201

Received April 2, 1969

Dilute protein concentrations are commonly measured by the

method of Lowry et al. (1). While this method is simple and sen-
sitive, it suffers from interference by many compounds. Bennett
(2) and Kuno and Kihara (3) use membrane filters to remove in-
terfering materials. This communication presents a simple and sen-
sitive procedure, not requiring membrane filters, for handling 20
to 40 protein samples simultaneously; it utilizes some features of
the filter paper assay of Mans and Novelli (4) and the dye method
of Kuno and Kihara (3).
Aliquots containing lo-200 pg protein are spotted on 2 x 2 cm
squares of filter paper (Whatman 42) and dried in a stream of
warm air. Many samples can be applied to a single piece of filter
paper previously marked into labeled squares with a soft pencil.
The thoroughly dry squares are cut apart and put into cold 7.5% tri-
chloroacetic acid to fix the proteins within the paper; 2 ml is used
for each square. The acid solution is then heated and maintained at
80°C for half an hour to hydrolyze and remove nonproteinaceous
material. The acid solution is then discarded; the papers are washed
with ether/ethanol (l/l v/v) and then ether to remove the tri-
chloroacetic acid and lipids and to dry the papers. Once thoroughly
dry, the papers can be stored for analysis.
Samples containing 5-25 pg protein are stained with xylene bril-
liant cyanin G (Michrome No. 1224)) 10 mg/ml in 7% acetic acid,
for 15 minutes at 50” with occasional swirling; 2 ml is used for
each paper. Excess dye is then removed with hot (50-60’) 7% ace-
tic acid until the background is almost white. The papers are then
drained dry and each paper is placed into a separate test tube. 5
1 This work was supported by NSF-URP grant GY-4654 and NSF research
grant GB-6226.

146
 DETERMINATION OF PROTEIN 147

ml of destain solution (66 ml methanol, 34 ml awater, 1 ml concen-

trated ammonia) is added to each tube. The dye released into the
solution is measured at 610 mp.
Samples containing 50-200 pg protein are stained with naph-
thalene blue black (Michrome No. 1113), 10 mg/ml in acetic acid/
methanol/water l/4/5 v/v/v at 50’ for 15 minutes. The papers are
rinsed with hot ‘7% acetic acid several times to remove excess dye.
The free liquid is discarded and the papers put into individual
tubes. 5 ml of 0.05 N NaOH is added to each tube and the dye
released measured at 620 rnp.
In both procedures standard and sample papers are treated to-
gether until the dyes are released from the papers.
The coefficient of variation within a test ranges from 0.02 to 0.05
for both procedures; the more accurate determinations are made
near the upper limit of each method. As the standard values vary
slightly from test to test, standards should be included in every
assay.
The protein values obtained by applying the two dye procedures
to the same leaf extracts agree with one another and with values
obtained by direct nesslerization of the Kjeldahl digests (5). The
xylene brilliant cyanin G method of protein determination is about
as sensitive as UV absorption measurement at 215 and 225 rnp (6)
and five times as sensitive as the naphthalene blue black CS method;
the latter is as sensitive as the Folin-Lowry procedure (Table 1).

TABLE 1
Relative Sensitivities of Three Methods for Determining Protein Concentration
Absorbance

Bovine albumin, pg

Method 10 20 30 60 100 150 200

Folin-Lowry (1) - - - 0.22 0.44 0.64 0.83

Naphthalene blue - - - 0.24 0.50 0.78 1.0
black
Xylene brilliant 0.29 0.63 0.90 - - - -
cyanin G t

The absorbance of the Folin-Lowry solutions was measured at 700 mp. The final
volume of each sample was 5 ml.

Reducing agents are often included in extraction media to obtain

more active enzyme preparations (7) and to increase the extractable
proteins (8). We have found that the addition of K&O, to Xan-
thium leaf extracts altered the Folin-Lowry protein values even
148 BRAMHALL ET AL.

when the proteins were precipitated immediately after extraction

(9). Extracts that were precipitated immediately after preparation
contained only 70% as much apparent Folin protein as extracts to
lwhich 0.01 M bisulfite was added; extracts made with the same con-
centration of reducing agent already in the homogenizing medium
contained more than twice as much apparent Folin protein as those
made without a reducing agent. These anomalous results may have
been caused by Folin reactive phenol& (10). The naphthalene blue
black test, on the other hand, did not respond to bisulfite additions;
it indicated that all samples contained the same amount of protein
(Table 2).

TABLE 2
Effect of K&O5 on Apparent Extractable Protein Determined
by Two Methods
Protein content, mg/ml
Naphthalene
Treatment blue black Folin-Lowry

Extract (pH 7.2 phosphate buffer) 2.5 1.4

Extract and 0.01 1M KISzOs 2.5 2.1
Extract made with buffer containing 0.01 M K&&05 2.5 3.2
2 gm (fresh weight) Xanthium leaves were extracted with 10 ml solution.

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1. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J.,
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2. BENNETT, T. P., Nature 213, 1131 (1967).
3. KUNO, H., AND KIHARA, H. K., Nature 215, 974 (1967).
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5. MINARI, O., AND ZILVERSMIT, D. B., Anal. Biochem. 6, 320 (1963).
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(1968).
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