VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

INTERNATIONAL UNIVERSITY

SCHOOL OF CHEMICAL AND ENVIRONMENTAL ENGINEERING

BIOCHEMISTRY LABORATORY

EXPERIMENT 1: QUANTITATIVE
DETERMINATION OF PROTEIN
CONCENTRATION IN SOLUTION BY
LOWRY ASSAY
Instructor: MSc. Ngo Nguyen Tien Dat

Group 03 – Section Thursday

Group 4 member

No. Student’s name Student’s ID Contribution

1 Trần Gia Linh BTCEIU21095 100%
2 La Nhật Khanh BTCEIU21091 100%
3 Nguyễn Trần Thảo Uyên BTCEIU21125 100%
4 Phạm Thị Hồng Ngọc BTCEIU21105 100%
5 Dương Hoàng Thảo Linh BTCEIU21094 100%

Date of submission: 28/11/2024

 Biochemistry Laboratory_Semester 1_Year 2024-2025

Table of Contents
I. ABSTRACT ........................................................................................ 2
II. INTRODUCTION...............................................................................2
III. MATERIAL AND METHOD............................................................ 3
 Materials:............................................................................................. 3
 Methods:...............................................................................................3
IV. RESULTS AND DISCUSSION........................................................4
 Result:...................................................................................................4
 Discussion:........................................................................................... 7
V. CONCLUSION....................................................................................8
VI. REFERENCES....................................................................................8

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I. ABSTRACT

The Lowry method is commonly used to quantify protein concentration in solutions.

Through the experiment, our group has understood how to apply the Lowry test in
quantitative analysis to determine protein concentration from solutions and UV-VIS
spectrometers. In addition, because the Lowry method has very high sensitivity, allowing the
detection of protein at very low concentrations (about 1 μg/ml), it can be seen that the Lowry
method is a reaction that mainly occurs with peptide bonds and aromatic amino acids
(tryptophan, tyrosine), so it is highly specific for proteins. Fortunately, our group has
successfully performed this experiment because the process is relatively simple and does not
require too complicated equipment. In addition, the Lowry method is very important in
medicine and is often used to determine protein concentration in serum samples, tissue fluids,
cells and purified proteins.

II. INTRODUCTION

An assay is a technique that quantitatively measures the presence or quantity or

functional activity of a target entity (analyte) which may be a drug or a biochemical or organic
sample. There are four types of assays including chemical assays, Immunoassays,
microbiological assays, bioassay. Assays are commonly used in laboratories such as
spectrophotometry, thin layer chromatography, column chromatography, etc [1]. In addition,
assays can also be used in drug discovery [2] and metal detection [3].

Some of the techniques for quantitatively measuring protein concentration in a

solution in biochemical research include Biuret, Lowry, Bradford, BCA, and
spectrophotometric assays. Each method has some limitations in sensitivity or is based on the
reaction of specific amino acids in the protein. In the four tests Biuret, Lowry, Bradford, and
BCA, chemical reagents are added to produce a color, and the intensity is measured
spectrophotometrically. In addition, a "protein standard" is also treated with the same reagents.

In this experiment, the Lowry assay is used to determine the amount of soybean
protein extract. The Lowry test was chosen for this class of experiments because it is highly
sensitive and widely used, and it is also capable of detecting protein levels as low as 5 ug. The
principle of color development of the Lowry test is coordination of peptide nitrogen atoms
with Cu2+ and the addition of a second reagent (Folin-Ciocalteu) to increase the amount of
color development. A standard curve is prepared with bovine serum albumin due to its low
cost, high purity and ready availability. The disadvantages of Lowry are poor specificity,

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many interfering substances (such as TRIS buffer, sucrose, ammonium sulfate, sulfate, phenol
and citric acid, etc.), the linear relationship of the standard curve is not really tight [4].

Figure 1: Reactions of the Lowry protein assay

Soybeans were used in this experiment because soy protein has advantages over other
natural proteins, namely low cost, non-animal origin, relatively long shelf life, as well as
stability. In addition, the ability to combine its properties with similarity to tissue components
and the ability to reduce the possibility of thermal degradation [5].

III. MATERIAL AND METHOD

 Material:
This experiment seeks to quantify the protein concentration in a solution with the
established Lowry method, pertaining to the apparatus and reagents listed below
Equipment:
 15 test tubes  1 100-1000 μL micropipette
 1 test tube rack  1 10 mL pipette
 1 100 mL Erlenmeyer flask  1 mortar and pestle
 1 100 mL beaker  1 spectrophotometer
 1 50 mL beaker  9 cuvettes
 1 50 mL volumetric flask  1 centrifuge tube
 1 50 mL graduated cylinder  1 centrifuge
Reagents:
 0.1% Albumin
 Folin reagent
 0.1N Sodium hydroxide (NaOH)
 Copper sulfate pentahydrate (CuSO4·5H2O)
 Sodium carbonate (Na2CO3)
 1% Sodium citrate
 Method:
First of all, the solution would be diluted in each test tube with the numbers 1 through
6 to create the standard albumin solution at six different concentrations. The volume of

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albumin and water was precisely placed into each test tube (1 to 6) immediately, following
this table.

Table 1: Standard solutions of albumin

Test tube 1 2 3 4 5 6
[Albumin] (µg/mL) 0 50.0 100.0 150.0 200.0 250.0
Volume of 0.1% Albumin (mL) 0 0.5 1.0 1.5 2.0 2.5
Volume of water (mL) 10.0 9.5 9.0 8.5 8.0 7.5
Moving to the next part, transferring protein solution from tubes 1 to 6 to 1' to 6'
respectively. Additionally, two more test tubes, 7' and 8', were prepared as duplicates of the
extracted and diluted protein solution. Solution C would be prepared by a mixture of A and B
at a ratio of 49:1. From that, 2.0 mL of solution C was added to each test tube (1' to 9'),
shaken thoroughly, and then left for ten minutes. Next, each test tube (1'-9') was filled with
0.2 mL of Folin reagent, well mixed, and let sit for ten minutes. Finally, filled each test tube
(1' to 9') with 2.4 mL of distilled water, give it a good shake, and wait another five minutes.

After thoroughly mixing and letting it stand for five minutes, tungsten blue was able to
obtain. Because each test tube had a different tungsten hue due to the varying amounts of
tyrosine and tryptophan, also noticed noticeable color changes, such as the BSA standard at
tube (1' to 6') (BSA stands for bovine serum albumin standard).

The solutions from the test tubes (1' to 9') would be put into the cuvette and the first
layer of distilled water would be added to the cuvette. Then carefully transferred the test tube
data to the cuvettes for measurement at a wavelength of 750 nm using a UV-VIS
spectrophotometer. (The absorbance measurements at 750 nm allow the determination of
unknown concentrations of proteins).

IV. RESULT AND DISCUSSION

 Result:

Figure 2: The contents of protein concentration in the cuvette from 1’ to 10’

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The cuvettes are arranged from left to right. The first cuvette serves as a blank for the
other cuvettes, which solely contain water with the solution C, and there is no protein present.
From the cuvette second to the cuvette tenth, the protein concentration presents the blue color.
However, the cuvettes 5' and 6' exhibit a slightly darker blue color, while the remaining three
cuvettes, 7', 8', and 9', display a lighter blue color.

Table 2: The concentration of albumin and absorbance of the standard solution at λ750nm

Tube
1’ 2’ 3’ 4’ 5’ 6’
number
[Albumin]
0 50 100 150 200 250
(µg/mL)
Absorbance
0.00 0.004 0.119 0.176 0.223 0.311
(A750)
The absorbance increases sharply through each test tube. The first test tube was used
as a blank, and it contained no protein concentration in the tube. Tube 6' yielded the highest
absorbance of 0.311, while tube 2' recorded the lowest absorbance of 0.004.

Figure 3: The standard curve of the absorbance with the concentration of albumin (µg/mL)

The standard curve yields an R-squared value of 0.9683. The more closely the R-
squared number approaches unity, the more accurately the linear model describes the
variations in absorbance. The absorbance at 0.004 with respect to 50 µg/mL seems far from
the trendline. It can be said that an outlier is an unexpected variable. Despite the high R-
squared value and its close to unity, the absorbance at 0.004 significantly differs from the
other absorbances. However, with only 0.176 nearly acceptable absorbance, there are no

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variables that fit the trendline. In addition to the R-squared value, the trendline provides the
regression model equation y = 0.0013x - 0.0232, where x represents the protein concentration
and y represents the measured response of absorbance for protein.

The diluted protein concentration was calculated from the standard curve, as shown in
Figure 3

𝑦 0.0232
With the provided linear equation 𝑦 = 0.0013𝑥 − 0.0232 ⇔ 𝑥 = 0.0013 + 0.0013

0.114 0.0232
The diluted protein concentration of tube 7’: 𝑥 = 0.0013 + 0.0013 = 106 µ𝑔/𝑚𝐿

0.119 0.0232
The diluted protein concentration of tube 8’: 𝑥 = + = 109 µ𝑔/𝑚𝐿
0.0013 0.0013

0.122 0.0232
The diluted protein concentration of tube 9’: 𝑥 = + = 112 µ𝑔/𝑚𝐿
0.0013 0.0013

The average absorbance calculated by

0.114+0.119+0.122
𝐴𝑑𝑖𝑙𝑢𝑡𝑒𝑑 = = 0.118
3

The standard deviation obtained

With sample diluted protein N = 3 and the mean of diluted protein 𝑋 = 0.118

2
∑𝑛𝑖=1 𝑋𝑖 − 𝑋 0.114 − 0.118 2 + 0.119 − 0.118 2 + 0.122 − 0.118 2
𝑆= = = 0.00406
𝑁−1 3−1

Table 3: Absorbance of the diluted protein solution at λ750nm

Diluted protein
Test tube
7’ 8’ 9’
[Diluted protein]
106 109 112
(µg/mL)
Absorbance 0.114 0.119 0.122
Average absorbance Adiluted = 0.118
Standard deviation 0.00406
Based on the regression model equation of the standard curve, the concentration of
diluted protein can be determined, which results in 106, 109, and 112 µg/mL with respect to
each test tube, 7’, 8', and 9’, respectively. The test tube 9' yielded the highest concentration of
diluted protein and absorbance. The average absorbance of 0.118 indicates that it is close to
the absorbance of tube 3', which falls within the range of 0.004 to 0.119, allowing the average

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concentration of diluted protein to be determined on the standard curve. Additionally, the

standard deviation of 0.00406 demonstrates that the Lowry assay technique for this
experiment's results is more repeatable and trustworthy, enabling the standard deviation to
assess the reliability and precision of the experimental result.

 Discussion:

There are a variety of assays that can be used to detect the presence of proteins in the
samples. Nevertheless, the Lowry assay is utilized in this experiment, which employs two
reagents to identify protein, such as Folin-Ciocalteu and hydroxyl copper. The coordination of
peptide bonds with alkaline copper and the reduction of the Folin-Ciocalteu reagent by
tyrosine and tryptophan residues in the protein are a result of the color change in protein
concentration from left to right, as demonstrated in Figure 2. Furthermore, the concentration
of the color protein in the sample will be indicative of its concentration upon reaction with the
Folin reagent. As the second reagent was introduced to the test tube with the aim to enhance
the level of color development. The copper in the Lowry solution can react with the peptide
bonds in the sample to create a peptide-copper bond [8]. Following reaction with hydroxide
ions in Folin's reagent, this bond facilitates the reduction of copper to copper (II). The copper
(II)-peptide complex has a blue color. Copper may attach to the four peptide bonds due to its
excess electrons and the presence of an empty orbital on the nitrogen-hydrogen (NH) of the
peptide bond [8]. The Folin's reagent can reduce copper since copper favors the +2-oxidation
state, allowing electrons to be readily extracted by even a weak base. [8]

Figure 3 illustrates that the obtained absorbance variable does not align with the
trendline, with the exception of a single variable at 0.176 that approaches the trendline. The
R-squared value of the Lowry assay trendline in the scientific paper closely resembles unity,
suggesting that the absorbance variable nearly aligns with the trendline [7]. Only a handful of
variables deviate significantly from the trendline [7], whereas the absorbance variables in this
experiment do not fit well with the trendline. The variable absorbance in this experiment can
be attributed to human-generated error. The error number in the standard curve can be
attributed to the pipetting process. During the experiment, using an inaccurate or inconsistent
micropipette to transfer the solution can lead to an error number when a small amount of
sample proteins is taken. Furthermore, incorrect use of the 10 ml pipette can lead to incorrect
reagent ratios, as it affects the measurement of the volume taken from the solution to dissolve
the sample protein.

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On the other hand, the results for the concentration and absorbance of diluted protein
are achieved with accuracy and precision. Table 3 illustrates that each tube's concentrated
diluted protein has a gap between three numbers, whereas the absorbance of the diluted
protein fluctuates between three and five. In addition to the concentration and absorbance of
diluted protein, a lower standard deviation indicates better reproducibility and precision in the
process of experimentation, such as pipetting and measurement.

Despite the perfect and accurate creation of the results, the trendline still lacks
precision. Replicating this experiment could aid students in refining their pipetting technique
for accurate sample or solution transfer. By refining these techniques, students can easily
achieve accuracy and precision results, thereby avoiding errors in the standard curve.

V. CONCLUSION

To sum up, the Lowry assay is used to quantify the amount of protein in a sample
through chemical reactions. The color of the sample will change depending on the
concentration of protein in the sample. A sample with a high protein concentration will have a
dark blue color and vice versa. In this experiment, the color change from light blue to dark
blue corresponding to the amount of standard albumin solution added to each test tube will be
observed. The result of the experiment is successful. During the experiment, it was shown that
from tube 1' to tube 6', the intensity of the blue color increased due to the different amounts of
protein in each tube. The more albumin solution was added to the test tube, the darker the blue
color. Tubes 7', 8', and 9' had a darker blue color than the remaining tubes when the diluted
protein solution was added. The Lowry assay is a simple technique and is often used in the
analysis of protein-containing samples because this method has high sensitivity, low cost, and
high accuracy. The Lowry assay is used in many fields such as clinical and industrial. Some of
the major applications are Protein purification and characterization, Enzyme kinetics studies,
Protein quantification in clinical samples, Monitoring protein expression levels in cell culture,
and Analysis of protein-protein interactions [6]. To achieve successful results, it is necessary
to pay attention to proper sample and reagent preparation. During the experiment, clean the
experimental equipment before experimenting, and check the spectrometer before measuring
the wavelength.

VI. REFERENCES

BIOCHEMISTRY LAB MANUAL (2024-2025)

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[1] Assays, types of assays, principle and prerequisites of assays and bioassay. SlideShare.
[Link]
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[2] The Importance of Assays in Drug Discovery and Development.

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[3] What Is an Assay and How Investors Think About It?. Investopedia.
[Link] (accessed 2024-11-27).

[4] Lowry method for quantitative detection of protein concentration. Medicilon.

[Link]
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[5] Soybean Protein - an overview | ScienceDirect Topics.

[Link]
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[6] Proteomics, C. Protocol for Lowry Protein Assay. Creative Proteomics.
[Link]
(accessed 2024-11-27).

[7] Deepachandi, B.; Weerasinghe, S.; Andrahennadi, T. P.; Karunaweera, N. D.;

Wickramarachchi, N.; Soysa, P.; Siriwardana, Y. Quantification of Soluble or Insoluble
Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to
Standard Lowry Assay. International Journal of Analytical Chemistry 2020, 2020, 1–8.
[Link]

[8] Foist, L. "Lowry Protein Assay: Principle, Protocol & Mechanism." [Link], [Link],
accessed November 27, 2024, [Link]
[Link].