ALGHAMDI, Amina Rose 9/05/19

CLAMOR, Raven 9/05/19

2018-03103
2018-02375

EXPERIMENTS 1 & 2
EXTRACTION AND ISOLATION OF PROTEINS
PROTEIN QUANTITATION THROUGH SPECTROPHOTOMETRIC METHODS

Background of the experiment

The experiments were done with the aim of isolating and quantifying proteins namely casein and
albumin from natural resources such as milk and egg, respectively. The methods and principles employed
in the processes involved were also explained and identified.

Results and Discussion

Proteins basically constitute the basis of living tissues in an organism. On the basis of mass, these
biomolecules are considered to be the major components of the dry weight of cells. In this experiment,
initial stages of protein characterization were expolored through the extraction, isolation, and quantitation
processes.
Table 1 and 2 show the appearance and amount of crude protein from the extraction and isolation
processes.

Table 1. Data from extraction of albumin from egg

Albumin from egg Centrifuge 1 Centrifuge 2 Centrifuge 3

Weight of 6.4789
centrifuge tube (g)

Weight of 7.24 7.04 6.97

centrifuge tube with
protein (g)
Weight of protein .7611 .5611 .4911
extract (g)

Color of protein off-white

extract

Table 2. Data from extraction of casein from milk

Casein from milk Centrifuge 1 Centrifuge 2

Weight of centrifuge 5.2646

tube (g)
Weight of centrifuge 6.1110 7.1559
tube with protein (g)
Weight of protein .8464 1.8913
extract (g)

Color of protein creamy white

extract

Figure 1 presents the standard calibration curve for the Bradford assay which was utilized in
obtaining the concentration of the proteins through their absorbance readings.

1.5
y = 0.7248x + 0.1676
R² = 0.6417

1.125
Absorbance

0.75

0.375

0
0 0.3 0.6 0.9 1.2
BSA Concentration

Figure 1. Calibration curve for Standard BSA

Table 3 presents the absorbance readings obtained for each of the protein solutions.

Table 3 . Absorbance Values for Protein Solutions (Group ○)

Albumin Casein

Trial 1 0.588 0.969

Trial 2 0.714 0.942

Trial 3 0.807 0.990

Average 0.703 0.967

Std. Dev. 0.110 0.024

RSD (ppt) 156.5 24.8

Gtab 1.1543 1.1543

Gexp (lowest) 1.046 0.227

Gexp (highest) 0.945 0.958

The concentration values were obtained by using the line of best fit from the standard calibration
curve and the absorbance readings of the proteins.

Table 4. Average Absorbance and Concentration Values of the Proteins (obtained from Absorbance)
Protein Albumin Casein

Ave. Absorbance 0.703 0.967

Ave. Concentration 0.739 1.103

In the experiments done, albumin and casein were the proteins of interest extracted from their
respective sources namely egg white and milk. These proteins were chosen as subjects for purification
due to their natural abundance in the said sources. Protein purification usually poses an accompanying
risk of product loss to some extent. Hence, an efficient set of methods is one with minimal steps that
results to a yield with a high degree of purity. Nonetheless, the selection of which set of methods to utilize
depends on the different physical and chemical attributes of the target protein. In particular, the solubility
and polarity of the protein must be considered as these influence the target protein’s activity to different
solvents and treatments.

Purification commenced through the process of homogenization which paved way for the
disruption of the cell membrane of the source in order to isolate the target protein. Homogenization via
mechanical disruption took place during filtration upon stirring and pressing of the egg.1 Afterwards, the
target proteins were subjected to centrifugation and fractional precipitation. Centrifugation allows the
sorting of particles in accordance with its size, shape, and density. This was done for the purpose of
filtering out the unwanted components such as nucleic acid and other broken cells, from the target protein.
A similar process is fractional precipitation - which relies on solubility in separating particles from the
solution. During the experiment, steps such as lowering the temperature, modifying the pH condition and
adding organic solvents and salts were all geared towards the process of fractional precipitation.2

As observed in the extraction of casein in milk, the pH of the solution was adjusted with HCl in
order to reach its isoelectric point - known as the process of isoelectric precipitation. In this condition,
precipitate formation becomes ideal due to a decrease in solubility between the protein and water which
would consequently result to an increase in the interaction between the protein molecules. Another
method which deals with protein solubility is the process of salting out. This was done upon adding
saturated NH42SO4 for the purpose of extracting albumin from the egg whites. The attraction between
the ions in the concentrated salt solution and the water molecules tend to induce an interaction between
the protein molecules, and consequently resulting to the formation of insoluble precipitates. A similar but
quite opposite process is called the salting-in effect; this utilizes dilute concentrations of salt which enable
the attraction between proteins to weaken.2 During the course of the experiments, a significant factor
which had to be considered was the maintenance of a low temperature. Exposure to high temperatures of
proteins would lead to disruption of its structure affecting its function and properties. Hence, the isolated
proteins had to be refrigerated upon storage.

The sources of protein isolates in the experiments were both from animals. Animal tissues possess
a relatively weak membrane which bears the target proteins. Hence, a simple mechanical disruption of the
cell membrane was utilized. Nonetheless, this may still vary depending on factors such as the hardness or
softness of the animal tissues involved and the presence of an extracellular matrix.3 On the other hand,
other living things such as microorganisms and plants bear cell walls which contain many interfering
agents. In this case, the enzymes are utilized in a process called enzymatic lysis in order to facilitate
digestion and stabilization of the cell membrane.3 A well-buffered environment should also be considered
in this condition so as to protect the proteins.

The isolated proteins were then subjected to quantitation through the Bradford assay. This assay
depends on the interaction between the Coomassie brilliant blue dye and the arginine and aromatic
residues in the protein. The dye tends to bind to these residues, causing the maximum absorption to shift
from 470nm to 595 nm. The Bradford assay tends to be a quick process; its sensitivity also tends to be
unaffected by the presence of reducing agents that may be in the buffer. However, in basic conditions and
in the presence of detergents, the dye’s ability to bind to the protein may be affected.

Protein quantitation does not follow a single assay for all sorts of protein. Methods may vary
depending on the properties of the target protein. There are other methods that may be used to quantify
proteins. Colorimetric methods such as the Lowry method and Bicinchoninic acid method (BCA. The
BCA assay utilizes the Biuret reaction, where in the Cu2+ ions are chelated by the protein backbone and
then reduced to Cu1+ ions. The BCA then reacts with the Cu1+ ions forming a purple product which
absorbs at 562nm.4 The BCA assay is not that sensitive with the types of amino acids in the protein due to
the peptide backbone being involved in the reaction. In Lowry method, the copper ions bind with the
Folin-Ciocalteu reagent to produce a blue solution that absorbs at 660 nm. However, numerous substances
that may be present in the samples can easily interfere with the Lowry analysis. Furthermore, color
development tends to be a slow process in this method so a precise reaction time and ideal temperatures
are a necessity. Similar with the BCA assay, is the Biuret method of protein analysis. Given basic
conditions, cupric ions form a complex with the peptides and the peptide bonds of proteins resulting to
the formation of a purple complex, which absorbs at 540nm. This assay is not quite sensitive with the
composition of the mixture and thus, is generally used.4 Another method for quantification is known as
protein mass spectrophotometry. This includes labelling one protein sample with a heavier isotope like
13C while a second sample is labelled with a lighter isotope like 12C. The proteins are mixed and the mass

difference due to the isotopes can give the ratio of two sample peak intensities via mass analyzer which
corresponds to the relative abundance ratio.5

Most if not all of the quantitation methods present to date do not address concerns, such as presence
and amount of certain amino acids and interfering agents, to a full extent. Hence, the results obtained
from the quantitation may not be deemed accurate to a full extent. In addition to the concerns in the assay
itself, variations or inaccuracy of the results may also stem from personal errors during the preparation of
the solutions and the samples. Improper loading of the sample to the spectrophotometer resulting to the
disruption of the path of the light may also lead to inaccurate results.
Summary, Conclusions, and Recommendations
Albumin from egg and casein from non-fat milk were extracted via homogenization and centrifugation.
Factors such as pH and temperature were controlled to achieve optimum state for extraction. The isolated
proteins were then subjected to quantitation through the Bradford assay. Absorbance was read with a
spectrophotometer at 595 nm. Results show that some samples deviate highly from the standard. Sources
of error may include inaccurate dilution.

References
[1] http://www-users.med.cornell.edu/~jawagne/proteins_%26_purification.html (accessed Sep 2, 2019)

[2] Campbell, M. K.; Farrell, S. O.; McDougal, O. M. Biochemistry; Cengage Learning Asia Pte Ltd:
Singapore, 2018.

[3] https://people.umass.edu/~mcclemen/581Proteins.html (accessed Sep 4, 2019)

[4] Compton, S J, and C G Jones. “Mechanism of Dye Response and Interference in the Bradford Protein
Assay.” Analytical biochemistry. U.S. National Library of Medicine, December 1985. https://
www.ncbi.nlm.nih.gov/pubmed/4096375.

[5] Waterborg, J H, and H R Matthews. “The Lowry Method for Protein Quantitation.” Methods in
molecular biology (Clifton, N.J.). U.S. National Library of Medicine, 1984. https://
www.ncbi.nlm.nih.gov/pubmed/20512668.
APPENDIX

CALCULATIONS

Protein Concentration

y=0.7248x+0.1676
Albumin Absorbance=0.703
Casein Absorbance=0.806

Albumin
0.703 = 0.7248x + 0.1676
x=0.881

Casein
0.806 = 0.7248x + 0.1676
x=0.954

Grubb’s Test (sample calculation)

g = |xi-x̃|/sx
g=|0.588-0.703|/0.110
g=1.046
Gexp<Gtab
Therefore, accept xi