INTERNATIONAL UNIVERSITY-HCMC UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

REPORT 1
Quantitative determination of protein concentration by Lowry assay
Instructor’s name: [Link] Hong Lan

Class: Practice in Biochemistry Date:13/12/2023

Group 1:
Student ID Declaration of
Full name
Contribution

1 Nguyễn Phương Quỳnh BTBTUN22061 Introduction

2 Trần Nguyễn Cát Tường BTBTIU22293 Procedures

3 Lê Hồng Vy BTBTIU22306 Results + Data analysis

4 Nguyễn Thị Linh Giang BTBTIU22315 Discussion

5 Lại Thúy Phương BTBTIU22295 Conclusion

Total score: _______/100

 I. INTRODUCTION
1. Objective
- To determine the concentration of extracted protein in the soybean by Lowry assay
- To estimate the amount of protein in the soybean by Lowry assay
2. Abstract
- In this experiment, the Hartee - Lowry assay is utilized for protein quantification, and
a calibration curve is created using a standard protein with a known concentration.
- To create a color that can be measured colorimetrically, protein solutions are mixed
with the Folin-Ciocalteu reagent.
- To improve color development, a reagent of different concentrations is mixed with
albumin solution, which serves as an appropriate standard.
3. Material

Equipment Chemicals

Albumin solution 0.1%: D issolve 0.1g of

albumin in water to make 100mL of
Test tubes
solution

Solution A: Dissolve 2g Na2CO3 in NaOH

Pipettes (1mL, 2mL, and 5mL) and pumps 0.1 N to make 100ml

Solution B: Dissolve 0.5 g of CuSO4.5H2O

in sodium citrate 1% to make 100ml
Volumetric flasks (50mL, 100mL)

Solution C: Mixture of A and B at a ratio

of 49:1. Solution C is prepared by pipetting
1.0mL of solution B and transferring it to a
Beakers (50mL, 100mL) 50mL volumetric flask. Solution A is then
added to the 50 mL level

Graduated cylinder (50mL) Folin – Ciocalteu reagent

Falcons (50mL)

Filter paper (11mm)

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 Spectrophotometer

Centrifuges

II. PROCEDURES
1. Sample preparation: put 5g pulverized soybeans into a stone mortar.
2. Extraction
• 1st time:
- Add a small amount of 40mL distilled water to stone mortar.
- Grind sample, then pour the remaining water into the mortar, continue to grind.
- Transfer the resulting extract solution to a beaker, leave the soybean grounds in the
mortar.
• 2nd time: Do the same as the 1st time with 30 mL distilled water.
• 3rd time: Do the same as the 1st time with 30 mL distilled water.
3. Centrifugation
- Transfer all extracted solution into centrifuge tubes.
- Centrifuge the tubes at a speed of 5000 rs/m for 10 minutes.
- Remove any film formed on the surface of the solution.
- Carefully decant the clear liquid into a clean 100 mL beaker.
4. Dilution
- Pipette 1mL of extracted protein solution into a 100mL volumetric flask.
- Fill the flask to the 100mL mark with distilled water (dilutes 100 times)
- Dissolve 1mL of the 100-diluted solution in 99mL of distilled water (dilutes 10,000
times)
5. Preparation of standard solutions
- Label six test tubes from 1 to 6.
- Dilute the 0.1% albumin solution to the desired concentration in each of 6 test tubes
that are listed in table 1.
- Shake the tubes well.
6. Preparation of protein solutions for absorbance measurement
− Label 10 new test tubes from 1' to 10'
− Transfer exactly 0.4 mL standard albumin solution from tube 1 to tube 1'. Repeat this
process for tubes 2 to 6.
− Tubes 7' and 8': add 0.4 mL extracted protein solution (diluted 100 times).
− Tubes 9' and 10': add 0.4 mL protein solution (diluted 10,000 times).
− Add 2.0 mL solution C to each test tube, shake well and allow to stand for 10 minutes.
− Add 0,2 mL Folin reagent to each test tube, shake well and allow to stand for 10
minutes.
− Add 2.4 mL distilled water to each test tube, shake well and allow to stand for 5 more
minutes.
− Measure the absorbance at 750 nm (A750) of these solutions.

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III. RESULT AND DATA ANALYSIS

1. Result table:

Table 1 - Result table

Tube number 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

0.1710 0.1660 0.1172 0.1033

OD 0.1158 0.1290 0.1668 0.1796 0.1953 0.2261
0.1685 0.1103

ΔOD 0 0.0132 0.0510 0.0638 0.0795 0.1103 0.1553 0.0971

Protein
concentration 0 50 100 150 200 250 X Y
(μg/mL)

2. Using Microsoft Excel to draw the standard curve:

The standard curve show the relationship between ΔOD and protein
concentration (µg/mL)
0,25

0,2
y = 0,0004x + 0,1143
0,15 R² = 0,979
ΔOD

0,1

0,05

0
0 50 100 150 200 250 300
Protein Concentration (µg/mL)

Figure 1 – The standard curve shows the relationship between ΔOD and protein
concentration (μg/mL)

3. Calculation:

- Using the obtained equation, the unknown concentration in test tubes 7’-8’ (100-fold
diluted) and 9’-10’ (10.000-fold diluted) can be calculated:

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 y = 0.0004x + 0.1143

which, y for ΔOD (optical density) value.

, x for protein concentration.

- With solution X, we have ΔOD7’-8’ = 0.1553

→ 0.1553 = 0.0004x + 0.1143

→ x = 102.5

→ Protein concentration of X is 102.5 (μg/mL) = 1.03 x 10-4 (g/mL)

- With solution Y, we have ΔOD9’-10’ = 0.1103

→ 0.1103 = 0.0004x + 0.1143

→ x = -10 (This result does not make sense, the value for protein concentration can never
be a negative number)

- Absorbance values of tube 10’ fall out of the range of the standard curve. → Rejected

- The protein co ncentration of 100-fold diluted sample solution is:

1.03 x 10-4 (g/mL)

- The protein co ncentration of 10.000-fold diluted sample solution is:

Concentration100-fold diluted /100= 1.03 x 10-4 x 10-2 = 1.03 x 10-6 (g/mL)

- The amount of soybean protein in 100mL of extracted sample solution:

mProtein = 100 x X = 100 x (1.03 x 10-4) = 1.03 x 10-2 (g)

- The grams of protein in 40mL soybean extract: (1.03 x 10-2) x 40 = 0.412(g)

0.412
- The ratio of protein in 5 grams of soybean is: = 0.0824 = 8.24%
5

→ There are approximately 8.24g of protein in 100g soybean.

IV. DISCUSSION

- The test tube numbers 7’, and 8’ are identical, so theoretically, they have the same OD.
However, in this experiment, OD is slightly different (test tube number 8’ is less than 7’).

→ The difference between these tubes can come from the amount of Albumin solution, which
differs slightly from each test tube.

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- The test tube numbers 9’, and 10’ are identical, so in theory, they have the same OD. However,
in this experiment, OD and is slightly different (test tube number 10’ is less than 9’).

→ The difference between these tubes can come from the difference in the amount of Albumin
solution and the re-made of the test tube number 9’.

- While preparing the test tubes from 1’ to 10’, test tubes 7’,8’, and 9’ may have different
protein concentrations as these test tubes are re-made.

- As tube 10’ falls out of the range of the standard curve, it would be rejected.

- The test tube number 6’ has the bluest color due to the high amount of Albumin solution
(2.5mL compared to 0.5mL, 1.0mL, 1.5mL, 2.0mL).

Figure 2 – Test tubes from 1’-10’ after adding reagent

[Link]:

After this experiment, we had an overview of how to quantitate the determination of protein
concentration by Lowry assay. There are two reactions used for developing the intensly blue
color in Lowry method:

- The first reaction was the coordination of peptide bonds with alkaline copper from a biuret
assay.

- The second reaction was the reduction of the Folin-Ciocalteu reagent by tyrosine and
tryptophan residues in protein.

There were some mistakes when we did this experiment, such as we added the wrong amount
of Albumin solution to each test tube and the re-made due to the different protein
concentrations of each tube. Although the result of the experiment is not the same as what we
predicted before, and through this experiment, we realized we needed to be more careful
when doing experiments and gained experience for the next practices.

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REFERENCE

- Biochemistry Lab Manual.