UNIVERSITI TEKNOLOGI MARA JENGKA, PAHANG

FACULTY OF APPLIED SCIENCES

Bachelor of Science (Hons.) Biology

BIOCHEMISTRY

BIO462

Practical 2: Protein Determination

No. Name Student ID

1. NURUL SYAFIKAH ATIRAH BINTI RAHIMI 2023644426

2. TUAN NORUL NAJWA BINTI TUAN 2023415044

ZAMZURI

3. SITI NORSYAHFIQAH BINTI LANA BUSU 2023479278

4. NOR AMIERA NABILA BINTI SUHAIMI 2023424256

5. SITI FATIMAH BINTI SALAHUDDIN 2023622968

Lecturer’s name: Muhammad Afif bin Yusof

Date of submission: 3/5/2024

INTRODUCTION

A spectrophotometer is a machine that can be used to measure the amount of light that is able to be
absorbed by the protein sample(Libretexts, 2023). It is able to operate by passing a beam of light
through the sample and measuring the intensity that is able to reach the detector. The beam of light
contains photons that will encounter an analyte molecule inside a cuvette and possibly be absorbed. The
analyte that absorbs protons reduces the number of them in a beam of light which reduces the intensity
of transmitted light beam to the detector. Since the detector absorbs protons, it does not show the
amount of light transmitted but instead the amount of light absorbed by the sample.

Using several sensitive protein assay techniques, the goal of this experiment is to determine the protein
concentration. First, one can measure the amount of protein present in a sample by applying the
Bradford method. How this protein assay operates is the idea that an intense blue hue changes from
brown when denatured protein binds to Coomassie Brilliant Blue dye in an acidic environment.

Apart from that, the biuret protein assay is another widely used technique in which an alkaline
copper-peptide bond reaction forms a complex known as the biuret complex. The test relies on a shift in
color to confirm the existence of proteins. The sample will become a different shade of blue if proteins
are found. If a high concentration of protein is found and measured using a spectrophotometer, the
complex that forms will yield a strong violet color.

Under alkaline circumstances, the Lowry technique can also be used to determine the total amount of
protein present in a sample. Though it requires additional steps and reagents, this method is derived
from the Biuret reaction. Lowry protein assay exhibits high sensitivity.The Folin-Ciocalteu reagent,
which interacts with copper ions through peptide bonds to produce a blueish color when absorbance is
measured at 750 nm, was used in the protein detection process.

For this experiment too, BSA solutions at 0.2, 0.4, 0.6, 0.8, 2, 4, 6 and 8 mg/mL in water, the former
four solutions are used for the Bradford method and the Lowry method while the former four are for the
Biuret method. It is a method to acquire the absorbance data and plot their data on separate graphs for
each essay and deionized water is also used at sample blank.

OBJECTIVE

I. To understand the use and how to operate a spectrophotometer

II. To estimate the amount of BSA solution using the Biuret method, Bradford method, and Lowry
method
MATERIALS

● Spectrophotometer
● Calibration cuvette
● Distilled water
● Measuring cylinder
● Test tubes
● Test tube rack
● BSA solution at 0.2,0.4,0.6 and 0.8
● Biuret reagent
● Bradford reagent
● Lowry reagents 1
● Lowry reagents 2
● Micropipette and Micropipette tips
● Stop watch

PROCEDURE

BIURET METHOD

1. 1.5 mL of Biuret reagent was added to 5 test tubes labeled 1, 2, 3, 4 and

BLANK
2. 1.0 mL of 2.0 mg/mL BSA solution was added to all the test tubes except
the one labeled BLANK
3. The mixtures are mixed and incubated at room temperature for 30 minutes
4. At 555nm, the OD was read
5. Repeat with 4, 6, 8 mg/mL BSA solutions

BRADFORD METHOD

1. 3.0 mL of Bradford reagent was added to 5 test tubes labeled 1, 2, 3, 4, and

BLANK
2. 0.6 mL of 2.0 mg/mL BSA solution was added to all the test tubes except
the one labeled BLANK
3. The mixtures are mixed and incubated at room temperature for 5 minutes
4. At 595nm, the OD was read
5. Repeat with 4, 6, and 8 mg/mL BSA solutions
LOWRY METHOD

1. 2.5 mL of Lowry reagent 1 mixed with 0.25 mL BSA solution was added to
5 test tubes labeled 1, 2, 3, 4, and only 2.5 mL of Lowry reagent 1 in the test
tube labeled BLANK
2. After 10 minutes, 0.25 mL of Lowry reagent 2 was added to all the test
tubes and mixed well
3. After 30 minutes, absorbance was measured at 750 nm

RESULTS

i) Biuret Method

Concentration OD OD OD Mean ± S.D

of BSA (Replicate 1) (Replicate 2) (Replicate 3)

Blank 0.001 0.002 0.002 0.002

2 mg/ml 0.193 0.192 0.192 0.192

4 mg/ml 0.203 0.202 0.202 0.202

6 mg/ml 0.219 0.220 0.219 0.219

8 mg/ml 0.325 0.326 0.327 0.326

ii) Bradford Method

Concentration OD OD OD Mean ± S.D

of BSA (Replicate 1) (Replicate 2) (Replicate 3)

blank 0.000 0.000 0.000 0.000

0.20 mg/ml 0.378 0.375 0.373 0.375

0.40 mg/ml 0.725 0.726 0.733 0.728

0.60 mg/ml 1.016 1.015 1.012 1.014

0.80 mg/ml 1.131 1.130 1.129 1.130

iii) Lowry Method

Concentration OD OD OD Mean ± S.D

of BSA (Replicate 1) (Replicate 2) (Replicate 3)

Blank 0.002 0.002 0.002 0.002

0.20 mg/ml 0.136 0.134 0.132 0.134

0.40 mg/ml 0.185 0.186 0.167 0.179

0.60 mg/ml 0.312 0.310 0.304 0.309

0.80 mg/ml 2.336 2.330 2.329 2.332

GRAPH

i) Biuret Method
ii) Bradford Method

iii) Lowry Method

EVALUATION OF EACH METHOD BY THE CRITERIA

1. Convenience (how easy is it to do?)

2. Sensitivity (how well does it detect small amounts of protein?)
3. Generality (how consistent are the results among different proteins?)
4. Linearity (does it give a straight line plot of absorbance vs protein?)

BIURET BRADFORD LOWRY

CONVENIENCE ● Quite ● Most convenient ● Fairly

convenient ● Sample convenient
● Involved a incubated for 5 ● Total of
30-minute minutes only. incubation time
process of is 40 minutes.
incubation

SENSITIVITY ● The least ● Has higher ● Fairly sensitive

sensitive method sensitivity than Biuret but
compared to compared to not as sensitive
Bradford and Biuret and Lowry as Bradford
Lowry. ● Can cause errors ● Highly sensitive
● Can detect up to as it is also to low protein
0 -1 mg amount sensitive to concentration
of protein interference ● Can detect up to
● High accuracy ● Can detect up to 0-0.1 mg amount
and does not 0-0.01 mg amount of protein
rely upon the of protein ● Partially reliant
composition of ● Highly dependent on the
amino acid on the composition of
composition of amino acid
amino acid

GENERALITY Consistent Low consistent High consistent

LINEARITY linear Linear linear

DISCUSSION

The determinations of protein concentration are based on the amount, nature of protein to be
analyzed, the presence of interfering substances, the sensitivity of the equipment and the method
used (Meyer, M. H., 2018). In this experiment, the protein in BSA concentration was determined by
using the 3 methods which are Biuret Method, Bradford method and Lowry method and employed
with a spectrophotometer. The graph absorbance against concentration for the 3 types of method
constructed as shown on the result section from the samples prepared.

The determination of protein is measured by the amount of light that the sample absorbs (Hartmann,
2018). The first method that was carried out is the Biuret method and tested with the 2,4,6,8 mg/ mL
concentration of BSA and the spectrophotometer average reading shows 0.192, 0.202, 0.219 and
0.326 respectively. The value of the absorbance slightly fluctuates and decreases. The blank solution
that contains only the Biuret reagent shows 0.002 on the spectrophotometer. The blank is put as it
acts as a reference. Cu 2+ ions that present in the Biuret reagent form a complex with peptide bonds
of the protein and it should be obtained with the most linear result. However, the color of the
complex might be somewhat different depending on the concentration of the protein.

The result indicates that the BSA concentration for 0.1, 0.2, 0.3 and 0.4 mg/mL when used the
Lowry method comes with values of the average absorbance of 0.134, 0.179, 0.309 and 2.332
respectively and the value of the blank is 0.002. The result was observed that the Lowry method is
more sensitive than the Biuret method. It is based on the reaction of Cu + produced by the oxidation
of peptide bonds with the Folin-Ciocalteu reagent.

The Bradford method used the BSA concentration with lower amount of concentration with 0.2, 0.4,
0.6, and 0.8 mg/mL and resulted with the average absorbance value of 0.375, 0.728, 1.014 and 1.130
respectively. The blank sample is 0.000. It shows that the lower the amount of concentration gives
the lower reading of the absorbance. The method is dependent on the amino acid composition of the
measured protein and it is more efficient than other methods as it involves fewer mixing steps, no
heating required and it gives a more stable colorimetric response. It is proved by comparing the
result from the 3 methods mentioned before; the graph by the Bradford method is directly
proportional with the absorbance against the concentration. There is no fluctuation value of the
absorbance when using the Bradford method compared with the Lowry and Biuret method.

A lot of factors can cause the error in the experiment and are classified based on the 2 types of error
which are random error and systematic error (Helmenstine, 2022). The spectrometer may not be
calibrated properly or it happened when pipetting the reagents and the dye which can cause problems
such as inaccurate mixing and addition of the solutions.

Spectrophotometer should be down to the zero point by the reagent blank since it can be a very big
factor for error in the experiment. Besides that,the random error can occur due to the personal error
for instance when the test tube used is not clean enough or due to the inaccurate amount of volume
of solution which causes inadequacy and slightly diluted the solution prepared.

CONCLUSION

In conclusion, the objectives of the experiment were achieved. The best method for protein
determination is the Bradford method as it is more convenient compared to the Biuret method and
Lowry method with high accuracy and shorter waiting time. The graph of absorbance against
concentration of BSA protein for the Bradford method is directly proportional with no fluctuations
shows that it is the most accurate reading by the spectrophotometer.

REFERENCE

1. Mæhre, H. K., Dalheim, L., Edvinsen, G. K., Elvevoll, E. O., & Jensen, I. J.
(2018). Protein Determination-Method Matters. Foods (Basel, Switzerland),
7(1), 5. https://doi.org/10.3390/foods7010005

2. Helmenstine, A. M. (2022). Random vs. Systematic Error. ThoughtCo.

Retrieved from
https://www.thoughtco.com/random-vs-systematic-error-4175358

3. Libretexts. (2023, February 13). 2.1.5: Spectrophotometry. Chemistry

LibreTexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistr
y_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Che
mistry)/Kinetics/02%3A_Reaction_Rates/2.01%3A_Experimental_Determi
nation_of_Kinetics/2.1.05%3A_Spectrophotometry