FACULTY OF APPLIED SCIENCES AND

COMPUTING KUALA LUMPUR CAMPUS

Bachelor of Science (Honours) in

Bioscience with Chemistry

Course

: BABS2433 Immunology

Title
: Preparation of Immunoglobin G (IgG) from
chicken serum.
Group

:1

Date

: 27th October 2015

Prepared by :
Name

Student ID

Chong Khai En

15WAU08702

Khoh Pui Mun

15WAU09813

Objective:
1. To isolate and partially purify Immunoglobin G (IgG) from whole
blood serum of chicken.
2. To understand the various techniques in the process of blood
serum preparation.
3. To compare the protein concentration and purity of crude serum,
crude albumin and immunoglobulin G (IgG).

Introduction:
In this experiment, chicken serum was used for preparation of
immunoglobulin G (IgG). Serum is the clear liquid that can be
separated from clotted blood. Serum differs from plasma, the liquid
portion of normal unclotted blood containing the red blood cells,
white blood cells and platelets. It is the clot that makes the
difference between serum and plasma. Since serum contains two
major types of protein which are globulins and albumins. These two
major types of protein are good source of immunoglobulins.
Especially serum IgG, as it is more abundant than IgM, IgA, IgD or
IgE. Three purification techniques are used to isolate Ig G from
serum such as salt precipitation, anion exchange chromatography
and dialysis.
In this experiment, sodium sulfate precipitation and dialysis
were used to isolate IgG from chicken serum. Sodium sulfate
precipitation is the common used technique for the preparation of
immunoglobulin from the whole chicken serum. 1.8g sodium
sulphate was added to the serum. Since the salt concentration of
the medium was increased, there was an interference with the
interaction of water molecules with charged polar groups on protein
molecules. The protein molecules became less hydrophilic
permitting greater hydrophobic interaction between protein
molecules. Eventually the proteins became insoluble. The salt
concentration differs at which each protein precipitates, with
overlap between similar proteins. When preparing the crude
immunoglobulin fractions, unwanted serum proteins stayed in
solution while immunoglobulins were precipitated out. The protein
concentration in the medium influenced the precipitation limits of
the protein and affected the coprecipitation limits of the protein.
Hence, when precipitating serum, sodium sulfate was added slowly
to avoid high local concentrations which would decrease the
specificity of the precipitation.

Dialysis tubing is also known as Visking tubing. It is a type of

semi-permeable membrane tubing used in separation techniques
that facilitates the removal or exchange of small molecules from
macromolecules in solution based on differential diffusion. In this
experiment, dialysis tubing is used to clean up the unwanted serum
proteins in the complex biological samples such as serum. Dialysis
separates proteins based on the size of the dialysis pores. From
diagram 1, the serum was put inside the dialysis tubing and tied a
knot at one end. It immersed in phosphate buffer saline. The large
IgG molecules could not pass through the dialysis pores, so it stayed
remain in the dialysis tubing. The small protein molecules could
pass through the dialysis pores, so it diffused out into phosphate
buffer saline. After one day, only pure IgG stayed remains in the
dialysis tubing.

Dialysis Membrane
Phosphate Buffer
Saline
Serum

(a) Before dialysis

equilibrium

(b) During

Diagram 1: The solution before and after dialysis.

Methodology:
Part I: Preparation of blood serum.
The fresh blood was allowed to stand at room temperature for 1
hour. The blood was stored overnight at 4oC to allow clot formation.
The serum was poured off and the majority of clot was remained in
bottom of flask. It was then centrifuged at 3000 rpm for 10 minutes.
The supernantant was transferred to new centrifuge tube and the
centrifugation was repeated.
Part II: Crude IgG preparation.
45 ml of chicken serum was preheated at 56oC for 20 minutes to
remove complement protein. The serum was centrifuged at 9000
rpm for 20 minutes at 4oC to remove supernatant, and then the

serum was filtered. 1.8g of sodium sulphate (18% w/v) was added,
mixed, swirled and dissolved. It was then centrifuged at 9000 rpm
for 10-15 minutes at 25oC. The pellet was dissolved in 5 ml distilled
water and thus became crude IgG. The dialysis tubing was wet with
distilled water. A knot was tied at one end, the 5 ml crude IgG was
lead in and then a knot was tied at opposite end. The dialysis tubing
was placed in 1-2 liter of phosphate PBS buffer and left at 4oC
overnight. The quantitative and qualitative measurements of crude
serum, crude albumin and partially purified IgG were done by using
Nanodrop spectrophotometer. Distilled water was used as a blank.
The absorption spectrums were printed out and analyzed.
Results:
1. Absorption spectrum of crude serum

2. Absorption spectrum of crude albumin

3. Absorption spectrum of partially purified immunoglobulin

G (IgG)

Result Analysis:

Sample
Crude serum
Crude albumin
Immunoglobulin G
(IgG)

Protein
concentration
(mg/ml)
31.194
28.801

A280
(nm)

A260/A28
0

31.194
28.801

0.65
0.89

2.684

2.684

0.58

Discussion:
Dialysis works by diffusion, which is a process results from the
random movement of molecules in solution and cause the net
movement from area of higher to lower concentration until
equilibrium is reached. In this process, unwanted molecules inside a
sample-chamber diffuse through a semi-permeable membrane into
another chamber of liquid. The large molecules cannot pass through
the pores of membrane, thus they will remain in the sample
chamber. On the other hand, the small molecules can diffuse across
the membrane and thus reach equilibrium across the whole solution;
therefore the concentration of those small molecules within the
sample is reduced.
Absorbance at 280 nm (A280) is used to determine the protein
concentration based on the absorbance of UV light by the aromatic
amino acids tryptophan and tyrosine, and by cystine, disulfide
bonded cysteine residues, in protein solutions. From the results we
obtained, the crude protein shows higher protein concentration than
IgG. Before dialysis, there are a lot of impurities inside the crude
protein especially salts and other contaminants. After dialysis was
done, some undesired contaminants including the salts were
removed, thus the IgG contains less contaminant. The protein
concentration also decreases because some tiny proteins which

smaller than the pore of dialysis tubing have been removed. This
can be seen from the A260/A280 ratio. In general, pure protein has
an A260/A280 ratio of 2.0. The crude protein should be has a higher
impurity than IgG, but our results show a different result. It may due
to some experimental errors.
Albumin presents more than half of all blood plasma proteins.
They are readily solubilize in water. The 18% w/v of sodium sulphate
was added to solubilize the albumin in the solution and become
supernatant after centrifugation. In addition, sodium sulphate also
allows the alpha-globulin (IgA) to dissolve in solution, while the
other proteins included gamma-globulin (IgG) was precipitated out.
In this way, IgG can be fractionated from each other by using the
solubility of proteins.
Dialysis membrane has different thickness and pore size.
Thicker membrane is tougher, but it will restrict solute flow and thus
equilibrium is reached more slowly. IgG is the predominant
immunoglobulin of internal components such as blood and
cerebrospinal fluid, which is around 80% of the total
immunuglobulins. It is also the smallest immunoglobulin, which has
a molecular weight of 150,000 Daltons. Therefore, it can readily
diffuse out of the bodys circulation into the tissues. The pore of the
tubing must be smaller than the size of IgG in order to hold it inside
the tubing. In contrast, IgM is a macroglobulin which is the largest
immunoglobulin, which has a molecular weight of 900,000 Daltons.
In other word, IgG can be hold in the tubing as well as other
immunoglobulin which are larger than IgG. As a result, the dialysate
is still containing other classes of immunoglobulin; therefore further
purification of IgG is needed.
Blood serum is blood plasma without fibrinogen or the other
clotting factors. It is clearer than plasma because of fewer proteins.
During the preparation of blood serum in this experiment, the blood
was stored overnight to allow clot formation as the diagram below.

Diagram 2: Serum and the clotted blood.

There are some protocols during the preparation of blood
serum in this experiment. The blood was stored at room
temperature which shielded from light. The cells should not be
refrigerated. The supernatant was transferred to centrifuge tube
carefully to avoid the disruption of cell layer or transfer any cells.
Some precautions must be carried out throughout the experiment to
obtain a more accurate result. A clean pipette was used and gloves
were worn to prepare blood serum to avoid contamination. The
dialysis tubing was clipped well on both ends to ensure that there is
no solution seeped through and thereby affecting the results.
Conclusion:
Dialysis is one of the purification techniques to isolate
immunoglobulin IgG from serum. Dialysis removes unwanted
contaminants and thus the partially purified IgG contains less
contaminant and lower protein concentration. All the samples are
not pure which have low value of A260/A280, this may due to some
experimental errors and therefore precautions are needed to obtain
a pure sample.

References:
Dialysis Methods for Protein Research, Thermo Fisher Scientific,
2015. Retrieved 15th November 2015 from:
https://www.thermofisher.com/my/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resource-

library/pierce-protein-methods/dialysis-methods-proteinresearch.html
Immunoglobulins Explained, by Dr. Guy Sherwood. Retrieved 15th
November 2015 from:
http://www.iwmf.com/sites/default/files/docs/ImmunoglobulinsExplai
ned.pdf
The use of sodium sulfates as the globulin precipitant in the
determination of proteins in blood, by Paul E. Howe, J. Biol. Chem.
1921, 49:93-107. Retrieved 15th November 2015 from:
http://www.jbc.org/content/49/1/93.full.pdf
Dialysis in Protein Research: Understanding the Basics, posted by
Protein Man on 28th May 2014. Retrieved 14th December 2015 from:
http://info.gbiosciences.com/blog/bid/197555/Dialysis-in-ProteinResearch-Understanding-the-Basics
Protocols for the Preparation of Blood Plasma and Serum,
ProImmune Limited, 2009. Retrieved 14th December 2015 from:
https://www.proimmune.com/ecommerce/pdf_files/PR31.pdf