BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012

Revised by Mary-Ann A. Landiao, v2016

Experiment 1

Isolation and Purification of Proteins

Score:

Name: Ana Margarita L. Baytion

Groupmates: Marla C. Basa
Joann H. Justiniane

Date Performed: June 28,

2016
Instructors Signature:

I. Objectives
1. The isolation of casein from milk by isoelectric precipitation
2. To isolate ovalbumin from egg white through salting out principle
II.Chemicals
10%

95% alcohol
III.

Hydrochloric

1% Silver Nitrate
Nitric Acid

acetone
1 N acetic acid
Buffered
ammonium
sulfate

acid

Apparatus/Materials/Equipment

Evaporated Milk

Stirring rod

hotplate

Pipet

Litmus Paper

250- mL beaker
IV. Diagram of the Procedure
Please write on a short bond paper.

Egg
Watch glass
Filter paper
25-mLGraduated
cylinder
Wash bottle

V. Summary of Theory
Milk contains 3.3% total protein where it is differentiated to three kinds:
caseins, lactalbumins, and lactoglobulins. Proteins from milk is accounted by 80
percent is casein (major protein) and 20 percent whey. Casein exists in milk as the
water soluble calcium salt of a phosphoprotein (phosphorous containing proteins) and
calcium caseinate. It is a mixture of alpha, beta and kappa caseins to form a cluster
called micelle. These micelles are responsible for the white opaque appearance of
milk. Casein can be isolated from milk by acidification to bring it to its isoelectric
point.
The pH value is known as the isoelectric point (IEP) of the protein and is
generally the pH at which the protein is least soluble in water at their isoelectric
points because they tend to aggregate by electrostatic interaction. At the isoelectric
point the number of positive charges on a protein is equal the number of negative
charges-neutral. Casein has the IEP of approximately 4.6 and it is the pH value at
which acid casein is precipitated. The positive end of one protein molecule attracts
the negative end of another protein molecule and the aggregates precipitate out of
solution. Since casein has a pH 4.6 so its net charge is zero, it is insoluble in

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

solutions of pH less than 4.6. The pH of milk is about 6.6; therefore casein has a
negative charge at this pH and is solubilized as a salt. If acid is added to milk, the
negative charges on the outer surface of the micelle are neutralized (the phosphate
groups are protonated) and the neutral water

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

insoluble protein precipitates (casein). The increased acidity causes the milk proteinscasein, to tangle into solid masses, or curds. It is important not to add too much acid,
because it may hydrolyze some of the lactose in the milk and reduce your yield.
To separate the fat from the protein in the casein precipitate, centrifuge the
milk to remove the fat and precipitate the casein micelles with low pH or precipitating
agents. The fat that precipitates along with casein can be removed by dissolving it in
alcohol since it can dissolve both oil soluble and water soluble compounds, for the
experiment we used in 95% alcohol.
Albumin is a type of protein that is soluble in water. Many types can be found
all over the natural world, and two of the most familiar examples can be found in egg
whites which is formally known as ovalbumin and in human. When combined with
water, such proteins form a colloid, a solution that appears homogeneous although it
actually contains multiple substances.
Ammonium sulfate is widely used for precipitation it acts by pulling water
molecules away from the non-polar units of proteins. The decrease in available water
molecules increases the surface tension and allows the protein to precipitate from a
solution. Further increase in the salt concentration implies that there is less and less
water available to solubilize protein, then protein starts to precipitate when there are
no sufficient water molecules to interact with protein molecules.
VI. Observations
Table VI.1 Isolation of Casein from Milk
Brand of Milk
Volume of Milk Used, mL
# of 10% HCl Drops Added
Weight of Watch glass + filter paper
# of Washings done before filtrate becomes Cl- free
# of HNO3 drops added
Weight of Watch glass + filter paper + Residue
Odor of the Residue
Description of Precipitate after air drying
Weight of Protein Isolated
Casein Yield (g/mL)

Alaska
12.5mL
12 drops
33.97g
1
2
35.10g
Rancid odor/sour odor
Dirty white color
1.13 g
1.904g/mL

Table VI.2 Isolation of Egg Albumin

Volume of Egg White Used, mL
Volume of (NH4)2SO4, mL
Describe mixture after 30 minutes
Describe the precipitate after centrifugation
Describe the mixture after 2 days
Description of Precipitate
Weight of Protein Isolated
Albumin Yield (g/mL)

50mL
50mL
Appearance of 3 layers
Slightly yellowish precipitate
White gob
White powder
2.9383g
0. 0588 g/mL

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

Table VI.3 First Weighing (Beaker w/ test tube)

Test tube
1.1
1.2
1
2
3
4
5
6

12.8039
12.6908
12.9572
12.7683
12.8952
12.8205

12.8040
12.6907
12.9573
12.7684
12.8953
12.8205

12.8041
12.6906
12.9572
12.7684
12.8953
12.8205

Table VI.4 Second weighing (Beaker w/ test tube)

Test tube
2.1
2.2
1
2
3
4
5
6

12.8040
12.6908
12.9575
12.7684
12.8952
12.8208

12.8042
12.6909
12.9576
12.7688
12.8953
12.8209

1.3

2.3
12.8043
12.6912
12.9577
12.7687
12.8954
12.8209

Average
weight
12.8040
12.6907
12.9572
12.7684
12.8953
12.8205
Average
weight
12.8040
12.6910
12.9576
12.7686
12.8953
12.8209

Constant weighing was used to produce data with less error since the initially
absorbed excess water from the test tubes and the samples and the moisture that
the samples can continually acquire when exposed to air that can add up to the net
calculated yield of the residue.
Table VI.5 Yield of Albumin
Test tube
1st weighing
2nd weighing
1
2
3
4
5
6

12.8040
12.6907
12.9572
12.7684
12.8953
12.8205

12.8040
12.6910
12.9576
12.7686
12.8953
12.8209
2.9685

Beaker + test
tube +residue
12.8377
12.7726
13.5584
13.1896
13.2663
14.2509

Yield of
albumin (g)
0.0337
0.0816
0.6012
0.4210
0.3710
1.430

VII.
Analysis (Incorporate data obtain in describing the process on isoelectric
isolation and purification of protein. Account for the losses of Casein and Albumin
during the process.)
The isoelectric point is significant in protein purification because it represents
the pH where solubility is typically minimal. The protein isoelectric point signifies
where the system has a zero net charge, the point where the protein will be
collected.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

Acidifying both milk and egg white lowers their pH value- both high source of
protein, causes the protein that needs to be isolated such as casein and albumin
reach their isoelectric point where in the desired isolation of protein can be obtained.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

VIII. Conclusion
Since casein exist in milk as calcium caseinate it was separated as an
insoluble precipitate by acidification of the milk to its isoelectric point (pH= 4.6) with
the calcium ions remaining in solution:
Acidifying milk essentially lowering its pH affects the milk by causing the
milk proteins, like casein, to unwind and unfold in a process known as protein
denaturing. It precipitates casein by coagulation (clotting) by increase
solubility of organic calcium and phosphorous in the micelle.
Milk has a pH of around 7 also make it an ideal growth material for
many microbes.
The
souring of
milk is a complex process
started by
the
action
of
microorganisms on the
principal
carbohydrate
in
milk,
lactose.
The
microorganisms
hydrolyze
the lactose into glucose
and
galactose. Once galactose
has been
formed, lactobacilli, a strain of bacteria present in milk, convert it to the sour-tasting
lactic acid. Since the production of the lactic acid also lowers the pH of the milk, the
milk clots when it sours due to the precipitation of casein.

To isolate albumin from eggs, the salting-out principle was used, it decreases
salt concentration, and the extent of isolating the albumin was to use its solubility
depending on their salt concentration in the solution, as well as the ionic strength
and temperature of the solvent used. Further increase in the salt concentration will
result in less water available to solubilize the protein. Having not enough water
molecules to interact with the proteins or the dehydration of the protein
environment, they precipitate and this is called salting-out.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

IX. References
R. Minard (Penn State Univ.) from Introduction to Organic Laboratory
Techniques: A
Microscale Approach, Pavia, Lampman, Kriz & Engel, Saunders,
1990. Revised 3/20/2000
Berg JM, Tymoczko JL, Stryer L. Biochemistry 5th edition: The Purification of
Proteins Is an Essential First Step in Understanding Their Function. New
York: W H Freeman; 2002.
Ritchie C.( 2 September
Purification.
Retrieved
Purification.html

2015;
from

original version: 2012-11-17). Protein

http://www.labome.com/method/Protein-

Nam Sun Wang. (n.d). ENZYME PURIFICATIONBY SALT (AMMONIUM SULFATE)

PRECIPITATION.
Retrieved
from
http://www.eng.umd.edu/~nsw/ench485/lab6a.htm
R. Denise (18 July 2012). ISOL ATION OF CASEIN FROM MILK AND
ACID/BASE
HYDROLYSIS
AND
NEUTRALIZATION.
Retrieved
from
https://www.scribd.com/doc/60232062/Isolation-of-Casein-From-Milk-and-Acid
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New
York; 2000.
M. Lomas (8 July 2014). Retrieved from https://www.quora.com/Why-does-milkspoil-so-quickly
P. Carpio (21 March 2011). Isolation and Characterization of Egg white
Proteins. Retrieved from https://www.scribd.com/doc/51216821/Isolation-andCharacterization-of-Egg-white-Proteins
J. Lawandi (27 August 2015). Retrieved from http://www.thekitchn.com/thescience-behind-why-acid-curdles-milk-222962
Mary McMahon (June 10, 2016). Retrieved from http://www.wisegeek.org/whatis-albumin.htm
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

Yeditepe University, Department of Chemical Engineering, Bioorganic

Chemistry Laboratory Manual, Istanbul; 2006

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.