BIOCHEMISTRY MANUAL

Prepared by Admer C. Daiblio, v2012

Experiment 1
Isolation and Purification of Proteins
The isolation of proteins from the chemical mass or biological fluid in
which they are founds requires normally the liberation of the cell contents by
disruption of the tissue in a suitable medium. The gentlest possible method for
breaking open the cell is generally desirable. Water, dilute salt, acid, or alkaline
solutions may be used to extract the proteins. Association of proteins with
organic substances may prevent the extraction of the desired protein with
aqueous solvents; this requires preliminary treatment of a ground tissue
suspension with weak detergent solutions or organic solvents such as acetone,
ether, or butanol.
Procedure
A. Isolation of Casein from Milk
1. Dilute an evaporated milk 1:1 or a skim milk 1:3 using tap water to make
23 mL solution. Indicate the brand of the milk on the data sheet.
2. Heat the milk to 40C then slowly add 10% HCl, dropwise with thorough
stirring. Do this over 10-minute period until a flocculent precipitate
forms. The pH must decrease to about 4.8. Avoid excess acid. Stir for 5
minutes. Centrifuge the mixture to allow the curd to settle, decant or
pipette out whey.
3. Pre-weigh a watch glass with a filter paper on it. Record measured weight
on the datasheet. Use ONLY this filter paper throughout all the filtrations to
be done, take good care not to tear/damage the filter paper to avoid loses.
4. Wash the curd by resuspending in 15 mL distilled water, stirring vigorously
and decanting. Filter and transfer the moist residue to a beaker.
5. Filter and repeat the washing until the filtrate is chloride free (test a portion
of the filtrate with 1% AgNO3; if filtrate turns yellow, chlorides are still
present). Note: Avoid losing some of the casein due to peptization while
washing repeatedly by adding 1-2 drops of dilute HNO 3 to wash water.
6. Let the curd dry on the filter paper. Repeat washing procedure, but his
time using enough 95% alcohol. Stir vigorously for 3 minutes. Filter again,
and remove the left alcohol by pressing the precipitate between filter
papers. Do this extraction step once again using acetone.
7. After filtration, remove left acetone by pressing precipitate between filter
paper. Open the filter paper and place on the pre-weighed watch glass.
Allow the acetone to evaporate spontaneously (air dry).
8. Weigh the watch glass with dried filter paper and residue on it. Determine
the weight of the dried crude extract and calculate Casein yield in g/mL.
B. Isolation of Egg Albumin
1. Carefully separate the yolks from two fresh eggs and get egg whites
including the chalazas. Discard the yolks. Measure the volume of the egg
white using a 250-mL beaker. Stir, and add 0.1 mL. 1 N acetic acid. Stir
gently.
2. Filter through chessecloth to break up the membranes. Stir vigorously with
a glass rod to speed up the passage through the cheesecloth. Place
filtrate in a 250-mL beaker. Add an equal volume (same volume with the
egg white used) of the buffered saturated ammonium sulfate. After 30
minutes, transfer solution to test tubes and centrifuge off the precipitate of
ovoglobulin. Ovomucin, glycoprotein responsible for most of the viscosity
of egg white will also be removed with these two steps.
3. Discard the supernate (liquid) and transfer the centrifugate (clear/yellow
precipitate) to a beaker. Add buffered ammonium sulfate slowly with
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.

stirring until the precipitate definitely ceases to dissolve. Stir the

opalescent solution occasionally. Refrigerate the mixture for 2-5 days.
4. Pre-weigh test tubes placed inside a beaker. These test tubes will be used
for the centrifugation of the refrigerated mixture.
5. Transfer refrigerated mixture to pre-weighed test tubes and centrifuge.
Decant all the supernate and discard.
6. Place test tubes back on the beaker previously used in the pre-weighing
and weigh. Determine the total weight of albumin isolated. Calculate for
Albumin yield in g/mL.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
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.

Revised by Mary-Ann A. Landiao, v2016

Score:

Experiment 1
Isolation and Purification of Proteins
Name: Marla C. Basa
Date Performed: June 23,
2016
Groupmates:
Instructors Signature:
Ana L. Baytion and Joann H.
Justiniane

Objectives:
1.) To know the principle of protein denaturing.
2.) To enhance our filtration knowledge and skills.
3.) To develop skills in isolating and purification proteins.

II

III

Chemicals:
Hydrochloric acid
Silver nitrate
Nitric acid

_10 %_
_1 %_
diluted

Acetic acid
1N
Ammonium sulfate saturated

Apparatus/Materials/Equipment:

The apparatus used in the experiment were 25mL graduated cylinder, 1mL
pipette, 250mL beaker, analytical balance, centrifuge, stirring rod, hot plate with
wire gauze, watch glass, wash bottle, aspirator, test tubes with cover, test tube
rack, test tube brush, thermometer and iron stand with iron ring and clamp. The
materials used were canned evaporated milk, two fresh eggs, cheesecloth and
filter paper.
IV

Schematic Diagram of the Procedure:

Please write on a short bond paper.

Summary of Theory:

Casein protein is one of the two proteins that make up dairy protein (the
other being Whey protein). It is typically known as the 'slow' digesting component
of Milk Protein. Between the curd and whey of milk, the curd is the Casein.

Casein exists in milk as the calcium salt, calcium caseinate. This salt has
a complex structure. It is composed of , , and caseins which form a micelle,
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.

or a solubilized unit. Neither the nor the casein is soluble in milk, singly or in
combination. If casein is added to either one, or to a combination of the two,
however, the result is a casein complex that is soluble owing to the formation of
the micelle.
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. For casein, the IEP is
approximately 4.6 and it is the pH value at which acid casein is precipitated. The
pH of milk is about 6.6; therefore casein has a negative charge at this pH and is
solubilized as a salt.
Acidifying milk essentially lowering its pH causes the milk proteins,
like casein, to unwind and unfold in a process known as protein denaturing.
The unfolded proteins are then free to interact with each other and clump
together in a way they could not do when they were properly folded. The milk
takes on a curdled appearance from the lumps of proteins that are binding one
another.
The casein micelles and fat globules behave as separate phases; they
prevent filtration of the milk and interfere with the usual separation methods. The
usual first step is to centrifuge the milk to remove the fat and precipitate the
casein micelles with low pH or precipitating agents.

Egg white albumin is also called ovalbumin and the major protein of egg
white. It is fractionated by means of ammonium sulfate precipitation.
The solubility of protein depends on the salt concentration in the solution.
At low concentrations, the presence of salt stabilizes the various charged groups
on a protein molecule, thus attracting protein into the solution and enhancing the
solubility of protein. This is commonly known as salting-in. However, as the salt
concentration is increased, a point of maximum protein solubility is usually
reached. Further increase in the salt concentration implies that there is less and
less water available to solubilize protein. Finally, protein starts to precipitate when
there are not sufficient water molecules to interact with protein molecules. This
phenomenon of protein precipitation in the presence of excess salt is known
as salting-out.
Many types of salts have been employed to effect protein separation and
purification through salting-out. Of these salts, ammonium sulfate has been the
most widely used chemical because it has high solubility and is relatively
inexpensive.
The centrifugate in procedure B.3 contains Albumin so the supernatant
should be discarded.

VI

Observations:

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.

A Isolation of Casein from Milk

Brand of Milk
Volume of Milk Used, mL
# of 10% HCl Drops Added
pH of the mixture
Weight of Watch glass + filter paper, g
# of Washings done before filtrate becomes
Cl- free
# of HNO3 drops added
Weight of Watch glass + filter paper +
Residue, g
Odor of the Residue
Description of Precipitate after air drying
Weight of Protein Isolated, g
Casien Yield (g/mL)

Alaska
12.5
12
4.75
33.97
1
2
35.10
rancid
Yellowish
1.13
0.0904

B 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)
VII

50
50
Separated into three layers
Whitish sticky
Formed two layers
Sticky, white
2.9375
0.05875

Results and Discussions

A. Casein Calculation
Casein isolated, g= (Weight of F.P + dried Casein + watch glass) (weight of F.P
+ watch glass) = 1.13
Since the volume of the milk used is 12.5mL;
Casein Yield, g/mL =

Test
tube
Number

= 0.0904

B. Albumin data and calculation

First Weighing, g

First
Reading
1
2
3
4
5

1.13 g
12.5 mL

12.8039
12.6908
12.9572
12.7683
12.8952

Second
Readin
g
12.8040
12.6907
12.9573
12.7684
12.8953

Third
Readin
g
12.8041
12.6906
12.9572
12.7683
12.8953

Averag
e
12.8040
12.6907
12.9572
12.7683
12.8953

Second Weighing, g

First
Readin
g
12.8040
12.6908
12.9575
12.7684
12.8952

Second
Readin
g
12.8042
12.6909
12.9576
12.7688
12.8953

Third
Readin
g
12.8043
12.6912
12.9577
12.7687
12.8954

Average

12.8042
12.6910
12.9576
12.7686
12.8953

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.

12.8205

12.8205

12.8205

12.8205

12.8208

12.8209

12.8209

12.8209

Since the results of the second weighing and first weighing are close ( 0.0004),
the second weighing will be used for the weight of the test tube initial.
Test tube number

Second Weighing,
g

1
2
3
4
5
6
Total Yield, g

12.8042
12.6910
12.9576
12.7686
12.8953
12.8209

Weight of the test

tube with dried
Albumin, g
12.8376
12.7726
13.5584
13.1896
13.2662
14.2508

Actual Yield, g

0.0333
0.0816
0.6008
0.4210
0.3709
1.4299
2.9375

Actual Yield = [(Weight of the test tube + dried Albumin) (Second weighing)]
= 2.9375 g
Albumin Yield, g/mL =
VIII.

2.9375 g
50 mL

= 0.05875

Analysis:

There is no pH at which ionic amino acids forms are absent, but there is a pH
at which there is an equal number of positive and negative charges present thus,
producing a no net charge situation. The no net charge pH value for an amino
acid solution is called its isoelectric point.
Casein has its isoelectric point at pH 4.6. Therefore, it is insoluble in solutions
that have a pH less than 4.6. When acid is added to milk, the negative charges
on the outer surface of the casein micelles are neutralized (by protonation of the
phosphate groups) and the neutral protein precipitates, with the calcium ions
remaining in solution:
Ca-caseinate + 2H+ casein + Ca2+
The Caseinate:

When the pH > pI, a protein has a net negative charge. The pH of the
mixture of the experiment is 4.75 making the yield less.
The isolation and purification of Albumin involves the method of saltingout, which is increasing salt concentration. However, according to Nam Sun
Wang in his article named Enzyme Purification by Salt (Ammonium Sulfate)
Precipitation an alternative method uses decreasing salt concentrations. In this
alternative method, protein is first precipitated with a concentrated salt solution.
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.

Then a series of cold (near 0C) ammonium sulfate solutions of decreasing

concentrations are employed to extract selectively the protein components that
are the most soluble at higher ammonium sulfate concentrations. The extracted
protein is recrystallized and thus recovered by gradually warming the cold
solution to room temperature. This method has the added advantages that the
extraction media may be buffered or stabilizing agents be added to retain the
maximum enzyme activity. The efficiency of recovery typically ranges from 30 to
90%, depending on the protein.
In the next experiment, it is better to apply this strategy suggested by
Jeanette Mowery and Lisa Seidman on their article Protein PurificationManual:
Purification of -galactosidase from E. Coli

IX.

Conclusion:

Therefore, we learned in this experiment that acidifying milk essentially

lowering its pH causes the milk proteins to undergo protein denaturing. When
an increase and then a rapid decrease in heat occurs, bacteria such as
lactococci and lactobacilli can form. Milk actually spoils when bacteria convert the
lactose into glucose and galactose, which results in the production of lactic acid.
Lactic acid produces casein and then forms a curd that can quickly allow the
entire amount of milk to curdle within 24 hours. Milk contains a variety of
nutrients like fat, carohydrate, protein & minerals and abundance of water which
provides excellent mobility to bacteria (inherent to milk as well as due to
contamination occurred during handling) and gives platform for enzymes activity.
The salt concentration at which a protein precipitates differs from one
protein to another. Hence, salting out can be used to fractionate proteins. The
principle of adding ammonium sulfate and centrifuging it is the formation of precipitate of
proteins that precipitates at certain percent and below and separating it to other proteins that
did not precipitate at such concentration via centrifugation. Salting-out process starts with the
determination of the percentage of ammonium sulfate of which the desired protein
precipitates the most. According to Chick and Martin in their journal article named The
Precipitation of Egg-Albumin by Ammonium Sulphate. A Contribution to the Theory of the
Salting-out of Proteins, egg albumin concentration in the filtrate is highest when
concentration of ammonium sulfate is 22% - 23% (w/v). Any increase in ammonium
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.

concentration will yield to the precipitation of the desired protein. The isolation of crude
Albumin is not accurate, the first addition of the ammonium sulfate should not be
equal to the egg white obtained from filtration and the mixture should be
refrigerated. The mixture in this experiment was placed in a twenty-four hours airconditioned room thus, less yield.
X.

References:
1.) vlab.amrita.edu,. (2011). Isoelectric Precipitation of Proteins: Casein from
Milk.
Retrieved
28
June
2016,
from
vlab.amrita.edu/?
sub=3&brch=63&sim=158&cnt=1
2.) Jakoby, W.B., Crystallization as a purification technique, Enzyme
Purification and Related Techniques, in Methods in Enzymology, Vol. 22,
Jakoby, W.B., Ed., Academic Press, 1971.
3.) Retrieved from:http://www.tacanow.org/family-resources/what-is-casein/
(June 28, 2016)
4.) Retrieved from:https://examine.com/supplements/casein-protein/ (June
28, 2016)
5.) Nam Sun Wang; Enzyme Purification By Ammoium Sulfate Precipitation
6.) Janice Lawandi, The Science of Why Acid Curdles Milk retrieved from:
http://www.thekitchn.com/the-science-behind-why-acid-curdles-milk222962
7.) Retrieved from: http://courses.chem.psu.edu/chem36/New%20Syn
%2036%20pdf/Exp112.pdf (June 28, 2016)
8.) Rahul Kumar, Dairy Technologist; Written: Feb 5, 2015
9.) Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/1429861 (June 28,
2016)

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.