ISOLATION OF PROTEINS: CASEIN AND ALBUMIN

Michael Jon P. Alvarez, Francesca Eirylle M. Arriola, Sieren B. Baguingan,

Pauline Anne E. Basco, Danielle Therese M. Bermudez and Leah Therese A. Camangon
Group 1 2B Medical Technology Biochemistry Laboratory

ABSTRACT
Selected proteins were isolated from their commercially available sources using different isolation techniques for each.
The group was assigned to isolate the proteins casein and albumin from a sample of skimmed or non-fat milk by
means of isoelectric precipitation and heat denaturation, respectively. The group used a 50 mL sample of Nestle nonfat milk which was first heated to 40 oC, after which 10% acetic acid was added dropwise until a curd-like precipitate
was formed, indicating that the isoelectric pH of casein has been reached. The precipitated casein was filtered by
gravity filtration and appeared as a white amorphous precipitate. The filtrate from which albumin was isolated
appeared as a cloudy yellowish liquid. It was placed in a 75 oC water bath. No precipitate was formed as the liquid
stayed in the water bath longer than the indicated 5 minutes.

INTRODUCTION
Milk is one of the substances that are abundant
in different types proteins. The primary protein
component of milk is a protein group called
caseins. Casein is the general term for a family of
related phosphoproteins. These make up about
80% of the proteins in cows milk and around 2045% of the proteins in human milk. The casein
family of proteins consist of several types of
casein, namely -s1, -s2, , and 6 [3]. These
form a multi-molecular granular structure called a
micelle, along with water and some salts [1].
They bear a specific amino acid composition that
is essential to for growth and development, which
makes milk one of the most essential
components of a healthy human diet. One of the
significant properties of caseins is that it
precipitates or coagulates at pH 4.6.
Aside from this, certain albumins can also be
found in milk in the form of lactalbumins.
Albumins are globular proteins that are soluble in
water and in dilute salt solutions and have the
ability to become denatured and coagulated by
heat application [2]. A specific example of a
lactalbumin is -lactalbumin, which is classified
as a whey protein. -Lactalbumin plays an
important role in the synthesis of lactose and is
essential in the process of milk synthesis [1].
Once casein has been isolated from the milk and
the milk has been made acidic, lactalbumin can
be precipitated out by heating the mixture.
In this experiment, several proteins were
isolated from their commercially available sources
which is needed in order to be able to study their
structure, properties, and different functions of
proteins. There are various methods by which
proteins can be isolated that involve different
principles. One of these methods is isoelectric
precipitation. The isoelectric pH (pI) is the pH
value at which the protein has zero net charge.
Isoelectric precipitation is done by adjusting the
pH of the protein source until the pI is reached.
This reduces the solubility of the protein, since it

is unable to interact with the other components

of its source, which will then lead to its
precipitation [1].
Another method by which proteins can be
isolated is by heat denaturation. Denaturation of
proteins involves the disruption of its secondary
and tertiary structure. Denaturation reactions,
however, are not strong enough to break the
peptide bonds that constitute to the proteins
primary structure, so the amino acid sequence
remains intact. Denaturation by heat application
can be used to disrupt hydrogen bonds and nonpolar hydrophobic interactions since heat
increases kinetic energy which causes intense,
rapid vibration of molecules, leading to the
breakage of the said bonds [4]. Other protein
isolation techniques or principles are salt-induced
precipitation wherein high concentration of salt
causes the solubility of the protein to decrease
greatly (a process called salting-out) [6], and
isolation by simple difference in solubility in
substances such as water.
The first part of this experiment aims to isolate
casein and albumin from skimmed milk by
isoelectric precipitation and heat denaturation
respectively, gluten from wheat flour by
difference in solubility and myoglobin from beef
heart by salt-induced precipitation.

EXPERIMENTAL
A. Sample used
Nestle Non-Fat Milk

B. Procedure
1. Isolation of casein from skimmed
milk
50 mL of the liquid milk sample was placed in a
100-mL beaker, and then heated on a hot plate
while maintaining the temperature at 40oC. Upon
reaching this temperature, 10% acetic acid was
added dropwise into the milk sample while the
solution was stirred gently after every 5 drops.
This was done continuously until the pH of the

solution reached 4.6, or when a precipitate or

curd was observed. The precipitated casein was
filtered out by gravity filtration and the filtrate
was set aside for the isolation of albumin.

2. Isolation of albumin from skimmed

milk
The filtrate which was previously obtained from
the filtration of casein was heated in a 75 oC water
bath for 5 minutes. After this, the liquid in the
solution was decantated off to obtain the
precipitated albumin.

The filtrate that the group obtained from

filtering out casein appeared as a slightly cloudy
yellowish liquid. After immersing it in a heated
water bath, the protein albumin was supposed to
be precipitated out of the solution. However, the
group failed to arrive at this result and it was
suspected to be due to the fact that the mixture
was overheated as it was left in the water bath
for over 5 minutes. The group proceeded to filter
the heated solution, retrieving only the same
yellowish liquid that was initially separated from
casein and heated.

RESULTS AND DISCUSSION

The group was able to obtain the following
results after following the procedure for isolating
casein and albumin from the milk sample:

2.
after
water

Table 1. Isolation of assigned proteins

PROTEIN
Casein
Albumin

DESCRIPTION
Clumps of yellowish white solid
precipitate (amorphous)
Yellow cloudy liquid

Upon addition of acetic acid to the milk sample,

the group was able to observe formation of curd.
As previously discussed, isolation of proteins via
isoelectric precipitation allows the protein to
precipitate out of its source or medium by
lessening its solubility once it reaches its pI. This
indicates that at the point wherein curd started to
form, a sufficient amount of acetic acid has been
added to adjust the pH of the sample to around
4.6 which is the pI of casein. After filtering the
solution, the collected casein precipitate was
observed to be a white amorphous precipitate.

of
the

Figure
Albumin filtrated
heating in
bath
The principle
behind the
separation
albumin involves

denaturation or the breaking of bonds of the

protein upon application of heat. When the
protein solution that came from the casein filtrate
was heated, the albumin was denatured and had
lost its secondary and tertiary structures. The
general process of denaturation can be explained
in the following diagram:
Figure 3. Effect of denaturation on the structure
of a protein
As a result of the alteration of its structure and

consequently some of its properties, the protein

loses its solubility in the solution, making it
possible for it to be isolated.
Figure 1. Filtered casein precipitate

REFERENCES
[1] Hurley, Walter L. Milk Composition: Proteins.
http://ansci.illinois.edu/static/ansc438/Milkco
mpsynth/milkcomp_protein.html 03/28/2016
[2] Minard, R. Isolation of Casein, Lactose and
Albumin
from
Milk.
http://courses.chem.psu.edu/chem36/New
%20Syn%2036%20pdf/Exp112.pdf
03/29/2016
[3] n.a. Milk Protein.
http://www.milkfacts.info/Milk
%20Composition/Protein.htm 03/29/2016
[4] Ophardt, Charles E. Denaturation of Proteins.
http://chemistry.elmhurst.edu/vchembook/56
8denaturation.html 03/28/2016
[5] Young, Derek R. Isoelectric Precipitation.
http://www.rpi.edu/dept/chem-eng/BiotechEnviron/PRECIP/precpph.html 03/28/2016
[6] Young, Derek R. Salt Induced Precipitation of
Proteins.
http://www.rpi.edu/dept/chemeng/Biotech-Environ/PRECIP/precpsalt.html
03/29/2016