BIO462

BIOCHEMISTRY

LAB REPORT 2 : PROTEIN

DETERMINATION

NAMES MATRIC NUMBERS

CORLAINE SHIERY ANAK JAMES 2023899958

FATIMAH BINTI HASRIN 2023491958

NURFARAH NABIHA BT AHMAD RASDAN 2023492024

NUR SHAZLIYANA BINTI HASRIN 2023638078

NUR FATIHAH BINTI MOHD RAKIDIN 2023414874

TITLE Protein Determination

INTRODUCTION A spectrophotometer is an instrument used to measure the amount of

light that a sample absorbs. This instrument operates by passing a
beam of light through a sample and measuring the intensity of light
reaching a detector.

The beam of (which is placed in the cuvette), there is a chance that

the analyte will absorb the photon. This absorption reduces the
number of photons in the beam of light, thereby reducing the intensity
of the light beam being transmitted out.

i. The intensity of light passing through a blank (I0) is measured.This

measurement is necessary, because the cell itself scatters some of
the light.

Intensity - the number of photons per second.

Blank - solution that is identical to the sample solution except that
the blank does not contain the solute that absorbs light.

ii. The intensity of light passing through the sample solution (I) is
measured. In practice, instruments measure the power rather than
the intensity of the light.

Power - energy per second, which is the product of the intensity

(photons per second and the energy per photon.

iii. Calculation involved two quantities: the transmittance (T) and the
absorbance (A)
T = I/I0
A = - log10 T

Transmittance - the fraction of light in the original beam that passes

through the sample and reaches the detector.
Absorbance - the fraction of the light absorbed by the sample.
A=1-T

However, a detector in a spectrophotometer will display the

absorbance reading rather than transmittance. Each unit in
absorbance corresponds with an order of magnitude in the fraction of
 transmitted light.

i. For A = 0, 100% of the light is transmitted and no light is absorbed.

ii. A = 1, 10% of the light is transmitted (T = 0.1) and 90% is
absorbed by the sample.
iii. For A = 2, 1% of the light is transmitted and 99% is absorbed.
iv. For A = 3, 0.1% of the light is transmitted and 99.9% is absorbed.

PRINCIPLE OF LIGHT ABSORPTION

i. Absorption of light is exponentially related to the number of the

absorbing solute that are encountered
(a)the concentration [c]
ii. Absorption of light exponentially related to the light path through
the absorbing solution.
Beer-Lambert relationship~Beer's Law
(a) usually expressed in term of the absorbance (A)
(b) The logarithm of the ratio of the incident light (I0) to the emergent
light (I)

BIURET METHOD

Under alkaline conditions copper reacts with the peptide bond to form
a purple complex (biuret complex) that is stabilized by the tetrate
molecule. Although relatively incentive, the technique is easy and
fairly consistent results with different proteins. Other amine
containing compounds and ammonium salts do interfere with this
analysis as do highly acidic solutions.

BRADFORD METHOD

The Bradford procedure is rapid and requires few pipetting steps.

The procedure does, however, suffer from inconsistency between
proteins and with many proteins the color yield is not linearly
dependent upon the amount of protein analyzed. The method is
based on the intense blue color that develops when the Coomassie
Brilliant Blue G-250 dye binds to an acid denatured protein.

OBJECTIVE ● To determine protein concentration in various types of protein

content.
● To determine protein concentration using three types of
protein assays which are Biuret, Bradford and Lowry assay.

MATERIALS ● Spectrophotometer
● Calibration cuvette
● Distilled water
METHODS Biuret Method

Reagent Preparation
1. Sodium potassium tartrate was placed into 1 liter volumetric
flask and was dissolved in about 400 mL of 0.2 M NaOH.
2. The mixture was stored in a plastic bottle.

Procedure
1. 1.5 ml of the Biuret reagent was added to each test tube.
2. 1.0 ml of the unknown or BSA was added to the test tube.
3. Each test tube was mixed and incubated at room temperature
for 30 minutes.
4. Read the OD at 555 nm.

Bradford Method

Reagent Preparation
1. An amount of 100mg of Coomassie Brilliant Blue G-250 was
dissolved in 50 mL of 95% ethanol.
2. 100 mL of 85% H3PO4 was added and mixed.
3. Diluted to a final volume of 1000 mL with water.

Procedure
1. 3.0 mL of the Bradford reagent was added to each test tube.
2. BSA with 4 different concentrations which are 0.1mg/ml, 0.2
mg/ml, 0.3 mg/ml and 0.4 mg/ml was diluted for 10% by
mixing 0.02 ml of BSA with 0.08 ml of distilled water.
3. Each test tube was mixed and incubated at room temperature
for 5 minutes.
4. Read the OD at 595 nm.

Lowry Method

Reagent Preparation
1. One volume of reagent B (0.5% copper sulfate pentahydrate,
1% sodium or potassium tartrate) was mixed with 50 volumes
of reagent A (2% sodium carbonate, 0.4% NaOH). This was
the mixture for Reagent 1.
2. Folin-Ciocalteu phenol reagent was mixed with an equal
volume of water. This is the mixture for Reagent 2.

Procedure
1. 0.25 ml of BSA was mixed with 2.5 ml of Lowry reagent 1.
2. 0.25 ml of Lowry reagent 2 was added and mixed well after
10 minutes.
3. The absorbance was measured at 750 nm after 30 minutes.

RESULTS BIURET METHOD

Blank= 0.023
Unknown=0.032

Concentrati OD OD OD Mean +-
on of BSA (replicate (replicate (replicate S.D
1) 2) 3)

2 mg/ml 0.210 0.201 0.207 0.206

4 mg/ml 0.310 0.317 0.316 0.314

6 mg/ml 0.343 0.342 0.348 0.344

8 mg/ml 0.383 0.383 0.387 0.384

BRADFORD METHOD

Blank=0.121
Unknown=1.865

Concentrati OD OD OD Mean +-
on of BSA (replicate (replicate (replicate S.D
1) 2) 3)

0.1 mg/ml 0.995 1.000 1.009 1.001

0.2 mg/ml 1.020 1.016 1.020 1.019

0.3 mg/ml 1.071 1.077 1.076 1.075

0.4 mg/ml 1.343 1.344 1.344 1.344

LOWRY METHOD

Blank=0.082
Unknown=0.208
Concentrati OD OD OD Mean +-
on of BSA (replicate (replicate (replicate S.D
1) 2) 3)

0.1 mg/ml 0.123 0.110 0.110 0.114

0.2 mg/ml 0.235 0.239 0.235 0.236

0.3 mg/ml 0.260 0.262 0.262 0.261

0.4 mg/ml 0.309 0.310 0.310 0.310

DISCUSSION EVALUATION OF EACH METHODS BY THE CRITERIA

Biuret Bradford Lowry

Convenience -good. -Excellent - Fairly

-very high -More convenient -
independent of convenient Can be
the than Lowry performed at
composition of method room
amino acids. - Dependent temperature -
-good general on the amino Double
protein assay acid incubation for
for batches of composition of 40 minutes.
material which the measured The first
yield is not a protein. incubation is
problem -Single 10 minutes,
-involve single incubation for second
incubation for 5 minutes incubation is
30 minutes. 30 minutes.

Sensitivity -least sensitive - More -Sensitive to

Bradford sensitive than low protein
method and Lowry method concentration -
Lowry method -0-0.01 mg -Sensitive over
-0-1 mg small small amount a wide range -
amount of of protein can -0-0.1 mg
protein can be be detected small amount
detected -Dependent on of protein can
the be detected
composition of
the amino acid

Generality The The The

consistency is consistency is consistency is
quite fair good excellent

Linearity Linear Non- linear Non-linear

concentration concentration
dependence dependence

Protein concentration determination based on the amount of protein,

the nature of protein analyzed, the presence of interfering
substances, the sensitivity of equipment, and the method used of
three protein assay methods which is Biuret, Bradford, and Lowry,
and was detected using a spectrophotometer. Spectrophotometer is
a device that measures the amount of light passing through a sample
and then measures the intensity of light from its detector, displaying
the absorbance reading rather than the transmittance.

By the experiment result, a graph of absorbance mean versus BSA

concentration were plotted for all methods. The Biuret method, BSA
concentration of 2,4,6,and 8 mg/mL used and a sample with an
unknown concentration. The absorbance obtained were 0.206, 0.314,
0.344 and 0.384 respectively. The reading repeated three times to
get an accurate result. A graph plotted with the mean obtained, we
were able to achieve a linear plot. The unknown concentration of the
sample was obtained from the graph.

The Bradford method used the BSA concentration with lower amount
of concentration with 0.1, 0.2, 0.3, and 0.4 mg/mL and resulted with
the average absorbance value of 1.001,1.019,1.075 and 1.3437
respectively.The blank sample is 0.078.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 as a 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.

The result indicated that the BSA concentration for 0.1, 0.2, 0.3, and
0.4 mg/mL when used the Lowry method comes with a value of the
average absorbance of 0.114, 0.236, 0.261 and 0.310 respectively.
The value of blank is 0.082. The value of absorbance decreases
slightly at concentration of 0.4 mg/mL. The result was to observe that
the Lowry method is more sensitive than the Biuret method. It is
based on Cu+ produced by oxidation of peptide bonds with the
Folin-Ciocalteu reagent.

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. 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.
CONCLUSIONS In conclusion, the Bradford technique appears to be the best method
in determining protein determination. The objectives of the
experiment to determine the protein by using Biuret, Lowry and
Bradford method by using the spectrophotometer was achieved.
Bradford method is the most convenient compared to Biuret and
Lowry method as Bradford method gives accurate reading, simple,
fast and high sensitivity. It also shows that the graph of the
absorbance is directly proportional against the BSA concentration
without any fluctuation value.

REFERENCES Which protein assay is best for you? (2021, August 10). Azure
Biosystems.
https://azurebiosystems.com/blog/protein-assay-best/

‌ uantifying proteins using the Bradford method. (n.d.).

Q
Www.qiagen.com.
https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/b
ench-guide/protein/protein-analysis/quantifying-proteins-using-the-br
adford-method