FMS 1 EXPERIMENT REPORT

PROTEOMIC SESSION

Name:

NAINA KARAMINA SAKINA

NIM :

07120100102

Group:

B2-3

FACULTY OF MEDICINE
UNIVERSITAS PELITA HARAPAN
2010

ABSTRACT
Protein is a crucial and essential component of all body tissues. Protein is crucial
and essential for body because it contains the essential amino acids that was
needed by bodies for their metabolic process. Protein also can help bodies to
generate such big number of new cells with their useful components, so no
wonder if the protein is often said as a crucial and essential component for the
body because every cell in the body is continuously being recycled or being
renewed in order to reach the homeostasis in the body. Because of the protein is
very important for the body, it also would be important to study about this amino
acids. We have to study about protein because we have to know what the protein
really is, what the source of protein, what are the components that exist in the
protein, and many more. One of the benefit of studying the protein is we can
know the advantages and the disadvantages of the protein. Because of that,
several experiments were done in this proteomic session in order to know more
about the proteins in the human body. Those experiments were used the blood
as their sample and they have several purposes, as to determine the
concentration of the protein in the sample, to determine the appropriate dilution
factor of the sample, to determine the molecular mass of certain proteins in total
serum protein, to determine the distribution of proteins among fractions, to get
the quantitative determination of the antigen AFP concentration in human serum,
and also to measure blood glucose level from serum sample. Those experiments
were essential because by doing those experiments we can know the amount of
the protein that exist in our body, so it can prevents from several disease that can
happen as a result of the deficiency or the excessive amount of the protein in the
body.

List of Figures
1.

Figure 1. Primary Protein Structure.....................................................7

1.

Figure 2. Secondary Protein Structure................................................7

1.

Figure 3. Hydrogen Bond in Tertiary Protein Structure........................8

1.

Figure 4. Tertiary Protein Structure......................................................8

1.

Figure 5. Quaternary Protein Structure................................................9

Contents
ABSTRACT........................................................................................................................ii
List of Figures....................................................................................................................iii
List of Tables......................................................................................................................iv
Contents...............................................................................................................................v
Chapter 1 INTRODUCTION..............................................................................................1
Chapter 2 MATERIALS AND METHODS........................................................................3
2.1. Materials...................................................................................................................3
2.1.1. Bradford Test.....................................................................................................3
2.1.2. Protein Separation Through SDS-PAGE...........................................................3
2.1.3. Enzyme-Linked Immunosorbent Assay.............................................................3
2.1.4. Colorimetric Determination of Blood Sugar Level...........................................4
2.2. Methods....................................................................................................................4
2.2.1. Bradford Test.....................................................................................................4
2.2.2. Protein Separation Through SDS-PAGE...........................................................5
2.2.3. Enzyme-Linked Immunosorbent Assay.............................................................6
2.2.4. Colorimetric Determination of Blood Sugar Level...........................................6
Chapter 3 RESULTS............................................................................................................8
3.1. Bradford Test............................................................................................................8
3.2. Protein Separation Through SDS-PAGE..................................................................8
3.3. Enzyme-Linked Immunosorbent Assay....................................................................9
3.4. Colorimetric Determination of Blood Sugar Level................................................10
Chapter 4 DISCUSSION...................................................................................................12
REFERENCES..................................................................................................................15

Chapter 1
INTRODUCTION
In the experiments in this proteomic session, the main substance that was used
is protein.
Protein is a large molecule composed of one or more chains of amino acids in a
specific order determined by the base sequence of nucleotides in the DNA
coding for the protein. Protein is something the body both is and cannot remain
without. Proteins are required for the structure, function, and regulation of the
body's cells, tissues, and organs. Each protein has unique functions. Proteins are
essential components of muscles, skin, bones and the body as a whole.
Examples of proteins include whole classes of important molecules, among them
enzymes , hormones, and antibodies. Protein is one of the three types of
nutrients used as energy sources by the body, the other two being carbohydrate
and fat. Proteins and carbohydrates each provide 4 calories of energy per gram,
while fats produce 9 calories per gram. The word "protein" was introduced into
science by the great Swedish physician and chemist Jns Jacob Berzelius
(1779-1848) who also determined the atomic and molecular weights of
thousands of substances, discovered several elements including selenium, first
isolated silicon and titanium, and created the present system of writing chemical
symbols and reactions. Protein is the main component of muscles, organs, and
glands. Every living cell and all body fluids, except bile and urine, contain protein.
The cells of muscles, tendons, and ligaments are maintained with protein.
Children and adolescents require protein for growth and development. 1
There are three different structures of protein which is the primary structure, the
secondary structure, the tertiary structure, and the quaternary structure.
1 http://www.medterms.com/script/main/art.asp?articlekey=6554

The primary structure of peptides and proteins refers to the linear number and
order of the amino acids present. The convention for the designation of the order
of amino acids is that the N-terminal end (i.e. the end bearing the residue with
the free -amino group) is to the left (and the number 1 amino acid) and the Cterminal end (i.e. the end with the residue containing a free -carboxyl group) is
to the right.

Figure 1
in

the

secondary

structure,

there

are

typical

shapes

that have been develop which is coils (an alpha helix) or folds (beta pleated
sheets)

Figure 2
The -Helix
The -helix is a common secondary structure encountered in proteins of the
globular class. The formation of the -helix is spontaneous and is stabilized by Hbonding between amide nitrogens and carbonyl carbons of peptide bonds
spaced four residues apart. This orientation of H-bonding produces a helical
coiling of the peptide backbone such that the R-groups lie on the exterior of the
helix and perpendicular to its axis.
-Sheets
-sheets are composed of 2 or more different regions of stretches of at least 5-10
amino acids. The folding and alignment of stretches of the polypeptide backbone
aside one another to form -sheets is stabilized by H-bonding between amide
nitrogens and carbonyl carbons. -sheets can be depicted in ball and stick format
or as ribbons in certain protein formats.
Some proteins contain an ordered organization of secondary structures that form
distinct functional domains or structural motifs. Examples include the helix-turnhelix domain of bacterial proteins that regulate transcription and the leucine
zipper, helix-loop-helix and zinc finger domains of eukaryotic transcriptional
regulators. These domains are termed super-secondary structures.
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units of a given protein. Included in this description is the spatial
relationship of different secondary structures to one another within a polypeptide
chain and how these secondary structures themselves fold into the threedimensional form of the protein. Secondary structures of proteins often constitute
distinct domains. Therefore, tertiary structure also describes the relationship of
different domains to one another within a protein. The interactions of different
domains is governed by several forces: These include hydrogen bonding,
hydrophobic interactions, electrostatic interactions and van der Waals forces.
7

Figure 3

Figure 4

The quaternary structure is the structure formed by monomer-monomer

interaction in an oligomeric protein. Oligomeric proteins are proteins with multiple
polypetide chains that are held in association by the same non-covalent forces
that stabilize the tertiary structures of proteins. Oligomeric proteins can be
composed of multiple identical polypeptide chains or multiple distinct polypeptide
chains. Proteins with identical subunits are termed homo-oligomers. Proteins
containing several distinct polypeptide chains are termed hetero-oligomers.
Hemoglobin, the oxygen carrying protein of the blood, have more than one string
of amino acids which are then hooked together in this structure. It contains two
and two subunits arranged with a quaternary structure in the form, 22.
Hemoglobin is, therefore, a hetero-oligomeric protein. 2

2 http://themedicalbiochemistrypage.org/protein-structure.html

Figure 5

The first experiment in this proteomic session is the Bradford test. The Bradford
protein assay or Bradford test is one of several simple methods commonly used
to determine the total protein concentration of a sample. The method is based on
the proportional binding of the dye Coomassie to proteins. Within the linear range
of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie
binds. Furthermore, the assay is colorimetric; as the protein concentration
increases, the color of the test sample becomes darker. Coomassie absorbs at
595 nm. The protein concentration of a test sample is determined by comparison
to that of a series of protein standards known to reproducibly exhibit a linear
absorbance profile in this assay.3
Although different protein standards can be used, the most widely protein which
is Bovine Serum Albumin (BSA) was used in this experiment.The purpose of this
experiment are to determine the concentration of the protein and the appropriate
dilution factor of the solution. The solution that was used in this experiment is the
serum from the blood sample. Serum is the component of the blood that have
similarity in composition with plasma, but lacks the coagulation factors. Serum
3 http://ww2.chemistry.gatech.edu

and plasma can be gotten from the centrifugation process of the blood sample
that is not been added with the anticoagulant. If this sample being mixed, only
serum separates with other components (plasma already mix with other
components).
Blood plasma is the yellowish liquid component of blood in which blood cells are
normally suspended and various substances like food, waste products,
hormones and gases remain in solution. It transports everything except fat
droplets and, in vertebrates, oxygen.
It makes up about 55 percent of the blood by volume and is composed primarily
of water and proteins.
It composed about 92 percent water with 6.5 percent proteins. The remaining
components are 0.8 percent salts, 0.6 percent lipids and 0.1 percent glucose.
Blood plasma contains salts, hormones, and proteins. There are two major
groups of protein in blood plasma, which is albumin and immunoglobulins.
Albumin has the purpose to keep blood from leaking out of blood vessels and to
bind certain hormones, while immunoglobins involved in actively defending the
body against invaders such as bacteri and fungi. It also help clot the blood. The
primary function of blood plasma is to serve as a reservoir for water. It supplies
water to cells that are dehydrated and also absorbs excess water from cells. 4

The second experiment that was done is Protein Separation Through SDSPAGE. This experiment has the purpose to determine the molecular mass of
certain proteins in total serum protein and the distribution of proteins among
fractions.
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a
very common method for separating protein according to their electrophoretic
4 http://www.ehow.com/about_5502288_blood-plasma-regulations.html

10

mobility by electrophoresis (migration of charged molecules in solution in

response to an electric field) uses a discontinuous polyacrylamide gel as a
support medium and Sodium Dodecyl Sulfate (SDS) to denature the protein.
SDS is an anionic detergent which denatures proteins by "wrapping around" the
polypeptide backbone and SDS binds to proteins fairly specifically in a mass ratio
of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in
proportion to its length, example: the denatured polypeptides become "rods" of
negative charge cloud with equal charge or charge densities per unit length. It is
usually necessary to reduce disulphide bridges in proteins before they adopt the
random-coil configuration necessary for separation by size. In separating and
stacking gel, we use TEMED (tetramethylethylenediamine) which its function as
a catalyst in the presence of free radical initiatiors to accelerate the
copolymerization of acrylamide and diacetone acrylamide into PAD. In denaturing
SDS-PAGE separations therefore, migration is determined not by intrinsic
electrical charge of the polypeptide, but by molecular weight. Determination of
Molecular Weight is done by SDS-PAGE of proteins of known molecular weight
along with the protein or nucleic acid to be characterised. A linear relationship
exists between the logarithm of the molecular weight of an SDS-denatured
polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of
the distance migrated by the molecule to that migrated by a marker dye-front. A
simple way of determining relative molecular weight by electrophoresis (Mr) is to
plot a standard curve of distance migrated vs. log10MW for known samples, and
read off the logMr of the sample after measuring distance migrated on the same
gel.5
in this second experiment, there are six steps that required in order to reach the
goal. The steps are preparation of gels, casting of separating gel, casting of
stacking gel, fractionation of serum protein by ethanol precipitation, preparation
of samples for SDS-PAGE, and staining and destaining the gel. In the fourth
step, the purpose of the step is to fractionate protein by precipitation.
5 http://www.mcb.uct.ac.za/sdspage.htm

11

Precipitation is widely used in downstream processing of biological products,

such as proteins. This unit operation serves to concentrate and fractionate the
target product from various contaminants. For example, in the biotechnology
industry protein precipitation is used to eliminate contaminants commonly
contained in blood. Academic research on protein precipitation explores new
protein precipitation methods. The underlying mechanism of precipitation is to
alter the solvation potential of the solvent and thus lower the solubility of the
solute by addition of a reagent.6
Precipitation can be done by using several solvents. One of the solvent that can
be used in the precipitation process is the organic solvents. In the second
experiment we used the ethanol which is the organic solvent for the precipitation
process.
Addition of miscible solvents such as ethanol or methanol to a solution may
cause proteins in the solution to precipitate. The solvation layer around the
protein will decrease as the organic solvent progressively displaces water from
the protein surface and binds it in hydration layers around the organic solvent
molecules. With smaller hydration layers, the proteins can aggregate by
attractive electrostatic and dipole forces. Important parameters to consider are
temperature, which should be less than 0 C to avoid denaturation, pH and
protein concentration in solution. Miscible organic solvents decrease the
dielectric constant of water, which in effect allows two proteins to come close
together. At the isoelectric point the relationship between the dielectric constant
and protein solubility is given by:

S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k

is a constant that relates to the dielectric constant of water. The Cohn process for
6 http://en.wikipedia.org/wiki/Protein_precipitation

12

plasma protein fractionation relies on solvent precipitation with ethanol to isolate

individual plasma proteins.
a clinical application for the use of methanol as a protein precipitating agent is in
the estimation of bilirubin.7

The third experiment that was done is the enzyme-linked immunosorbent assay
which has the purpose to get the quantitative determination of the antigen AFP
concentration in human serum.
The last experiment in this proteomic session is the colorimetric determination of
blood sugar level. This experiment was done in order to measure the blood
glucose level from serum sample.
Blood glucose, is the bodys fuel that feeds the brain, nervous system, and
tissues. A healthy body makes glucose not only from ingested carbohydrates, but
also from proteins and fats, and would not be able to function without it.
Maintaining a balanced blood glucose level is essential to a bodys everyday
performance. Glucose is absorbed directly into the bloodstream from the
intestine and results in a rapid increase in the blood glucose level. The pancreas
releases insulin, a natural hormone, to prevent blood glucose levels from
excessively elevating, and aids in the moving of glucose into the cells. Glucose is
then carried to each cell, providing them with the energy needed to carry out its
specific function. Healthy blood glucose levels are considered to be in the 70-120
range. One high or low reading does not always indicate a problem, but the
glucose level should be monitored for 10-14 days. There are several different
tests that can be administered to determine whether an individual has a problem
maintaining a normal glucose level such as: a fasting blood sugar test, an oral
glucose test, or a random blood sugar test. 8 In this experiment, the blood glucose
7 http://en.wikipedia.org/wiki/Protein_precipitation
8 http://www.wisegeek.com/what-is-blood-sugar.htm
13

assay that was done is the assay by using the colorimeter or spectrophotometer.
A spectrophotometer or colorimeter makes use of the transmission of light
through a solution to determine the concentration of a solute within the solution. A
spectrophtometer differs from a colorimeter in the manner in which light is
separated into its component wavelengths. A spectrophotometer uses a prism to
separate light and a colorimeter uses filters. Both are based on a simple design
of passing light of a known wavelength through a sample and measuring the
amount of light energy that is transmitted. This is accomplished by placing a
photocell on the other side of the sample. All molecules absorb radiant energy at
one wavelength of another. Those that absorb energy from within the visible
spectrum are known as pigments. Proteins and nucleic acids absorb light in the
ultraviolet range. The following figure demonstrates the radiant energy spectrum
with an indication of molecules which absorb in various regions of that spectrum.
The design of the single beam spectrophotometer involves a light source, a
prism, a sample holder and a photocell. Connected to each are the appropriate
electrical or mechanical systems to control the illuminating intensity, the
wavelength, and for conversion of energy received at the photocell into a voltage
fluctuation. The voltage fluctuation is then displayed on a meter scale, is
displayed digitally, or is recorded via connection to a computer for later
investigation.
Spectrophotometers are useful because of the relation of intensity of color in a
sample and its relation to the amount of solute within the sample. For example, if
you use a solution of red food coloring in water, and measure the amount of blue
light absorbed when it passes through the solution, a measureable voltage
fluctuation can be induced in a photocell on the opposite side. If now the solution
of red dye is diluted in half by the addition of water, the color will be
approximately 1/2 as intense and the voltage generated on the photocell will be
approximately half as great. Thus, there is a relationship between the voltage

14

and the amount of dye in the sample.

Given the geometry of a spectrophotometer, what is actually measured at the
photocell is the amount of light energy which arrives at the cell. The voltage
meter is reading the amount of light transmitted to the photocell. Light
transmission is not a linear function, but is rather an exponential function. That is
why the solution was approximately half as intense when viewed in its diluted
form.
We can however monitor the transmission level and convert it to a percentage of
the amount transmitted when no dye is present. Thus, if 1/2 the light is
transmitted, we can say that the solution has a 50% Transmittance. Note that it is
always relative to a solution containing no dye. Transmittance is the relative
percent of light passed through the sample. What makes all of this easy to use,
however, is the conversion of that information from a percent transmittance to an
inverse log function known as the Absorbance (or Optical Density). 9

9 http://homepages.gac.edu/~cellab/appds/appd-g.html

15

Chapter 2
MATERIALS AND METHODS
2.1. Materials
2.1.1. Bradford Test
1. Bovine Serum Albumin Standard Set (0.125, 0.25, 0.5, 0.75, 1, 1.5, 2
mg/ml)
2. 1X Dye Reagent Vol. 1 ml
3. Cuvettes

2.1.2. Protein Separation Through SDS-PAGE

1. Laemmli sample buffer
2. Coomassie blue staining solution
50% Methanol
0.05% (v/v) Coomassie brilliant blue R-250
10% (v/v) Acetic Acid
40% H2O
3. TEMED

2.1.3. Enzyme-Linked Immunosorbent Assay

1. Rabbit anti-human AFP coated microtitter plate with 96 wells
2. Zero buffer (13 ml)
3. Reference standard set contain 0, 5, 20, 50, 150, and 300 mg/ml AFP,
4.
5.
6.
7.

lyophilized
18 ml enzyme conjugate reagent
11 ml TMB reagent
11 ml 1N HCl stop solution
Distilled water

16

8. Microtiter plate reader with a bandwidth of 10 nm or less and an optical

density range of 0-2 OD or greater at 450 nm wavelength.

2.1.4. Colorimetric Determination Of Blood Sugar Level

1.
2.
3.
4.
5.
6.
7.
8.
9.

Otoluidine reagent
Standard glucose solution 200mg/dL
Distilled water
Centrifuge
Spectophotometer
Cuvette
Reaction tube
Aluminium foil
Water bath

2.2. Methods
2.2.1. Bradford Test
20 l of each standard and unknown sample was pipetted into microcentrifuge
tubes and each tube was added by 1 ml of 1X Dye reagent. Blank sample should
be made using distilled water and dye reagent. The tubes were inverted in order
to mix the solution. After that, the tubes were being incubated at room
temperature for at least 5 minutes, it shouldnt be incubated longer than 1 hour at
room temperature. Then, the spectrophotometer was set to 595 nm. The blank
sample was used to zero the instrument and the absorbance of the standards
and unknown samples were measured by using the spectrophotometer.
2.2.2. Protein Separation Through SDS-PAGE
2.2.2.1. Preparation of gels
a 12% separating gel was prepared by adding 1.28 ml of Distilled water, 1.6 ml of
30% Acrylamide, 1.04 ml of 1.5 M Tris-HCL Buffer (pH 8.8), and 40 l of 10%
SDS to a 50 ml beaker. For the stacking gel, a stacking gel was prepared by
adding 0.7 ml of Distilled water, 0.165 ml of 30% Acrylamide, 0.125 ml of 0.5 M
Tris-HCL Buffer (pH 6.8), and 10 l of 10% SDS to a 50 ml beaker.
2.2.2.2. Casting of separating gel
17

The gel casting mould was assembled as instructed by the demonstrator. 80 l of

10% APS solution and 8 l of TEMED was added and swirled very gently for
about 5 seconds. About 3.6 ml of separating gel solution was added carefully to
avoid any spillages by using 1 ml automatic pipette. After that, the separating gel
solution was overlayed immediately with distilled water. Then, the separating gel
solution was left to polymerize for about 30 minutes and after it has been
polymerized, the distilled water was poured out.
2.2.2.3. Casting of stacking gel
20 l of 10% APS solution and 2 l of TEMED was added and swirled very gently
for about 5 seconds. About 1 ml of stacking gel was also added on top of
polymerized separating gel by using 1 ml automatic pipette and any bubbles
were tried to be avoided. After that, the comb was placed between the glass
plates with one end higher than the other and it was pressed down carefully so
that the teeth are level about 5 mm from the top of the separating gel. If
necessary, more stacking gel might be added to ensure that the sides of the well
are complete. Then, the separating gel was left to polymerize for about 20-30
minutes, the comb was removed carefully and the wells were being washed out
for at least 3 times with distilled water.
2.2.2.4. Fractionation of serum protein by ethanol precipitation
125 l of ice-cold ethanol and 250 l of serum were added to the 1.5 ml
microcentrifuge tube and the tube was vortex and stored in ice bucket. After it
was stored in ice bucket for 5 minutes, it was centrifuged for 3 minutes at 7000
rpm and 4C. After that, the supernatant was decanted carefully to another
microcentrifuge tube, it was labeled Es and stored in the ice bucket. A towel
paper was twisted and inserted into the microcentrifuge to draw off the last of the
supernatant from above the precipitated proteins by capillary action. Then, 1 ml
of chilled 50% ethanol was added to the precipitate to washed out the last traces
of supernatant. The samples were resuspended by repeated aspiration with 1000
l automatic pipette to break up the pellet (this step should tried to be done
quickly so that the preparation didnt heat up with the warmth of our fingers). 200
l of the samples was taken from the previous step and added to a new tube.

18

Then, the tube was centrifuged again for 3 minutes at 7000 rpm and 4C, the
wash supernatant was decanted into the plastic waste cup and the last drop was
removed with a twisted towel paper. The Ep fraction was the precipitated one. In
the end, the samples were stored in ice bucket.
2.2.2.5. Preparation of samples for SDS-PAGE
10 l of Es was taken out to another microcentrifuge tube and 45 l of sample
buffer was added to it. After that, 90 l of sample buffer was added to the Ep
fraction then it was vortex to mix the precipitate thoroughly with the sample
buffer. The 1000 l automatic pipette could be use to break up the pellet if there
was trouble resuspending the pellet. Then, Es and Ep tubes were put into a rack
in the boiling water and they were boiled for 5 minutes (only the tips of the tubes
need to be in contact with the boiling water). Next, the microcentrifuge tubes
were removed from the boiling water bath and the exteriors were dried with a
towel paper. The tubes then were centrifuged at room temperature for 3 minutes
to pellet any insoluble materials that may have precipitated during boiling. After
the centrifugation process was done, the samples were ready to be loaded into
the appropriate wells of the gel. About 5-12 l of the samples was loaded to the
gel. The precipitates shouldnt been stirred up before loading.
2.2.2.6. Staining and destaining the gel
The gel was removed and stained with coomassie blue in wobble table for 1
hour. After that, the gel was destained with destain solution in wobble table for 1
hour and the destaining solution was changed with fresh one every 1 hour until
background is clear. Then, the gel was wrapped in a piece of cellophane plastic.
2.2.3. Enzyme-Linked Immunosorbent Assay
The desired number of coated wells in the holder was secured. 20 l of standard,
specimens, and controls were dispensed into appropriate wells. After that, 100 l
of zero buffers was also dispensed into each well and it was mixed thoroughly for
30 seconds (it was very important to have complete mixing in this setup). Then, it
was incubated at room temperature (18-25C) for 30 minutes. After the
incubation process was done, the incubation mixture was removed by flicking

19

plate content into a waste container. Then, the microtiter wells were rinsed and
flicked 5 times with distilled water. The wells were sharply struck onto absorbent
paper or paper towel to remove all residual water droplets. 150 l of Enzyme
Conjugate Reagent was dispensed into each well and gently mixed for 5
seconds. Next, it was incubated at room temperature for 30 minutes. After the
incubation process was done, the incubation mixture was removed by flicking
plate contents into a waste container. Then, the microtiter wells were rinsed and
flicked 5 times with distilled water and they were sharply struck onto absorbent
paper to remove residual water droplets. 100 l of TMB Reagent was dispensed
into each well and gently mixed for 5 seconds. It was incubated at room
temperature for 20 minutes. After the incubation process was done, 100 l of
Stop Solution was added to each well to stop the reaction. Then, gently mixed for
30 seconds (make sure that all the blue color changes to yellow color
completely). The optical density was read at 450 nm with a microtiter reader
within 15 minutes.
2.2.4. Colorimetric Determination Of Blood Sugar

2.2.4.1. Performing standar curve standard glucose solutions

The glucose standars 500 mg/dl was provided. After that, the concentration
glucose standard point was being prepared. In this experiment the tube that was
used is tube number 3 which the concentration was 100 mg/dl, 0.5 ml glucose
standar, 0.5 ml water, and 1 ml of total ml per tube. After it has been prepared, 2
ml of 1% O-toluidine was added to it and well mixed. Then, the tubes were put in
a boiling water bath for 10 minutes. Last, measured at absorbance 630 nm.
2.2.4.2. Measuring of blood glucose
The blood that was drawn from a vein was transferred into a centrifuge tube.
After that, the blood sample was centrifuged. The centrifugation process obtained
serum. After the centrifugation process was done, the glucose concentration in

20

the provided serum sample of patient 1, 2 or 3 was determined using the Otoluidine method. 0.05 ml of distilled water (blank) or standard glucose (standard)
or serum (test) was added in a clean dry test tube, followed by adding 1 ml of Otoluidine reagent. Then, the content of each tube was mixed. The tube opening
was covered with aluminium foil. After the tube opening has been covered, the
tubes were put in a boiling water bath for 10 minutes. Next, test tubes were
removed from the water bath and cool under tap water. Last, the absorbance was
read at Imax 630 nm and the concentration of Blood Sugar Level (BSL) in the
provided blood samples was calculated using the absorbance reading of
standard glucose.

21

Chapter 3
RESULTS

3.1. Bradford Test

In this experiment, the standard protein which is Bovine Serum Albumin (BSA)
was used to be the compound for comparing the binding of it to a dye with the
binding of sample protein to a dye. There were five different concentration of BSA
that used in this experiment (one group made one protein standards). After
protein standards were made, we got the protein standards absorbance which is
shown in the table below:

Absorbance at 595 nm

0,172
0,28
0,489
0,639
0,892

0,173
0,278
0,49
0,646
0,89

BSA concentration (mg/mL)

Average
X
0,1725
0,279
0,4895
0,6425
0,891

Y
0,125
0,25
0,5
0,75
1

Table 1. Data for absorbances measurement and protein standard (Bovine

Serum Albumin) concentration.

Then, we made the standard curve based on the protein standards absorbance
which is shown in the figure below:

22

Standard Curve for Bradford Test

1.2
1
0.8

f(x) = 1.24x - 0.09

R = 1

0.6
0.4
0.2
0
0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Figure 7. Standard Curve for Bradford Test

From the above standard curve, we got the formula for measuring the
concentration of protein in our samples which is y = 1,2447x 0,091. The
meaning of the statement R2= 0,99531 is the linear shape was valid or can be
used. The data of our samples absorbance and the calculation of the protein
concentration in our samples is shown in the above table:

Sample 1

Mean (x)*

0.504

0.511

0.5075

II

1.235

1.247

1.241

NOTE:
Sample I : Tiara

Sample II : Adi

Table 2. Data of absorbances measurement of the samples.

The calculation of protein concentration is:
Formula y = 1,2447x 0.091 ; *( x = mean of the absorbance)

Tiaras sample:

Adis sample:
23

y= 1,2447(0.5075) - 0,091

y = 1,2447(1.241) - 0,091

= 0.63133-0.091

= 1.543804-0.091

= 0.54033

= 1.452804

Because we have diluted the sample with the comparison of 1:100, so the
resulted concentration above must be multiplied by the dilution factor (100) to get
the real concentration of protein in serum.
So, Tiaras protein concentration =

Adis protein concentration =

y= 0.54033 x 100

y= 1.452804 x 100

= 54 mg/ml

= 145.28 mg/ml

3.2. Protein Separation Through SDS-PAGE

NOTE: Es Adis

Figure 7. The gel

Ep Adis
M DNA marker
Es Tiaras
Ep Tiaras

24

The image above shown that well 1 was loaded with Ep from Tiaras sample, well
4 was loaded with Es from Tiaras sample, well 5 was loaded with DNA marker,
well 6 was loaded with Ep from Adis sample, and well 8 was loaded with Es from
Adis sample. The length of the gel (L) was 4 cm, and the distance between the
first line/top part and the last line/bottom part (d) of the chosen band was 2.1 cm.
To measured the standard curve, the DNA marker have to be visible in order to
know the size of the band. But in our gel result, the DNA marker was not shown.
So we had to use DNA marker from other group (B2-4) for measuring the
standard curve, which is shown in the figure of the gel result above:

The above figure shows that the DNA marker that was shown up were 75, 50, 37,
and 25 kdalton. The length of the maximum protein running in the gel (L) was 4
cm. The distance for each marker band is shown in the below table:
Size

d (cm)

(dalton)
75.000
50.000
25.000

0.7
1
2.1

Table 3. Data of specific sizes distances

25

After that, we have to know the data of the axis y = log of size of the band and
the axis x = Rf (Retention factor) which can be got from the formula: d/L (distance
per length). The result of the calculation and the data that we got is shown in the
below table:
Size
(dalton)
75.000
50.000
25.000
Length= 4 cm

Log of Size
(y)
4,88
4,70
4,40

d (cm)

Rf (x)

0.7
1
2.1

0,175
0,25
0,525

Table 4. Data for axis y (log of size) and x (Rf).

26

Then, we made the standard curve for SDS-PAGE based on log of size and Rf
that we got from the previous step. The standard curve is shown in the above
figure:

Standard Curve for SDS-PAGE

5.00

Log of Size

4.50

f(x) = - 1.29x + 5.07

R = 0.97

4.00
0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55
Rf

Figure 10. Standard Curve for SDS-Page

From the above standard curve, we got the formula for measuring the molecular
mass of the protein in bands in SDS-PAGE, which is y = -1.2907x + 5.066.
Before we calculate the molecular mass of the protein, we have to know the Rf
(Retention factor) first. The band that we used was the band with the 2.1 cm of
the distance (d = 2.1 cm). So the Rf from that band is :
Rf (x) = d/L = 2.1/4 = 0.525
And the calculation of the molecular mass of the protein in band is:
y = -1.2907x + 5.066
= -1.2907 (0.525) + 5.066
= - 0.684071 + 5.066
= 4.381929 (log of size)
The calculation above resulting the size which is log -1 4.381929
If we antilog the size resulting the different value of the size which is 23,988.33.
So it can be concluded that the molecular mass for the protein band is 23,988.33
dalton.

27

3.3. Enzyme-Linked Immunosorbent Assay

Standar

Absorbance

d
(nm)
1
0,041
2
0,086
3
0,156
4
0,388
5
0,8
Table 5. Data of Standard set AFP

Concentration(ng/ml)
0
5
20
50
150

Standard Curve for ELISA

200
Concentration (ng/mL)

f(x) = 196.21x - 12.72

R = 0.98

100
0
0

0.2 0.4 0.6 0.8

Absorbance (nm)

Figure11. Standard Curve for ELISA test.

From the standard curve in the above figure, we got the formula for measuring
the antigen AFP concentration. The formula is y = 196.2x 12.72 ( y protein
concentration ; x absorbance ).
To calculate the antigen AFP concentration using the formula, the absorbance of
the sample must be known. The absorbance of each sample is shown in the
below table:
Sample
1
2

Absorbance (nm)
0.020
0.033

The calculation of antigen AFP concentration in serum are:

Sample 1:

28

y = 196.2x 12.72
= 196.2(0.020)-12.72
= |-8.796|
= 8.80 ng/ml
Sample 2:
y = 196.2x 12.72
= 196.2(0.033)-12.72
= |-6.2454|
= 6.25 ng/ml
So it can be concluded that the antigen AFP concentration in sample 1 is 8.80
ng/ml and the antigen AFP concentration in sample 2 is 6.25 ng/ml.

3.4. Colorimetric Determination Of Blood Sugar Level

The glucose standard was used to determine the standard glucose absorbance.
The different concentration of glucose standard were used by each group (our
group used 100 mg/dl), so it resulting the variety of standard absorbance. The
standard glucose absorbance and its concentration are shown in the below table:
Standar

Result Result Mean

d
1
2
3
4

1
0.214
0.331
0.346
0.456

2
0.216
0.331
0.347
0.458

Absorbance
0,215
0,331
0,347
0,457

Concentration
60
80
100
120

Table 6. Data for standard glucose absorbance and its concentration

Then we made the standard curve of the standard glucose solution based on the
data of the above table which is shown in the below figure:

29

Standard Curve for BSL

150
100
conc. (mg/dL)

f(x) = 252.22x + 4.88

R = 0.94

50
0
0.2

0.25

0.3

0.35

0.4

0.45

0.5

A (nm)

Figure 12. Standard curve of Standard Glucose Solutions.

From the above standard curve figure, we got the formula for measuring the
blood sugar level in the samples. The formula is y = 252.22x + 4.8764. The
meaning of the statement R2= 0,99531 is the linear shape was valid or can be
used. To calculate the blood sugar level in the samples, the absorbance (x) of
each sample also have to be known.
The absorbance of each sample is shown in the below table:
Sample
1
2

Absorbance (nm)
0.2625
0.3625

The calculation of blood sugar level in the serum samples are:

Tiaras sample:
y = 252.22x + 4.8764
y = 252.22 (0.2625) + 4.8764
= 71.08 mg/dl
Adis sample:
y = 252.22x + 4.8764
y = 252.22 (0.3265) + 4.8764
= 87.23 mg/dl

30

So it can be concluded that the blood sugar level in Tiaras serum is 71.08
mg/dl and the blood sugar level in Adis serum is 87.23 mg/dl.

31

Chapter 4
DISCUSSION

The first experiment that was done in this proteomic session was the Bradford
test. The Bradford test is some method to determining the total serum protein
that is mostly consist of albumin and globulin. Total serum protein is the total
amount of protein that is found in blood. This experiment was also done to
determine the appropriate dilution factor of the solution. The process in this
experiment was depend on quantitating the binding of dye to an unknown protein
and comparing this binding to that different amount of a standard protein. The
standard protein that was used in this experiment is the Bovine Serum Albumin.
This standard have to be diluted in order to adjust the protein concentration, so
that it falls in standard range. To measuring the protein concentration in the
samples, the absorbance of the standards and samples have to be known. So
the spectrophotometer was used at 595 nm to measured the absorbance of
them. The spectrophotometer that was used in this experiment have to be set at
595 nm because the absorbance is in the optimum point for the form of binding
dye to protein. Optimum means the absorbance was at the peak and cannot go
up any further. After the absorbance of protein standards and samples was
determined, we got the formula for measuring the protein concentration in
samples serum protein by made a standard curve. The formula that we got for
measuring the protein concentration was y = 1,2447x 0,091. From the
calculation of protein concentration using the formula that we got from the
standard curve and multipled it with 100 (because the experiment required 100
times of dilution), we got different result of the total serum protein in each
sample. Tiaras total serum protein concentration is 54 mg/ml and Adis total
serum protein concentration is 145.28 mg/ml. These results showed the
abnormal amount of total serum protein concentration in both samples. Because

32

the normal total serum proteins in healthy adults are 55 90 mg/ml. So the
results of both samples were said to be abnormal because in Tiaras sample the
concentration was below the normal range, on the other hand Adis sample
concentration was above the normal range. The abnormal results that can be
known by comparing the results with the normal range of healthy adults might be
happened because of the human error in the process of mixing materials,
pipetting the volume of unknown sample or dye reagent, and other possible
errors. Because a deficiency (< 20 l) or excessive (> 20 l ) amount of the
samples or dye reagent could cause an increased and decreased in number of
total serum. The abnormal results also can be happened because the total
protein in the samples were low.
Because of many proteins that have difference in size and shape

33

have nearly identical charge and mass ratios, the second experiment in this
proteomic session which is the protein separation through SDS-PAGE can be
used to determine the molecular mass of protein in the total serum protein of the
samples. This experiment can determined the molecular mass of protein in the
total serum protein because this experiment was used the electrophoresis
process which is the process of separating macromolecules in an electric field.
SDS-PAGE was the method that used in this experiment. This method used a
discontinuous polyacrylamide gel as a support medium and sodium dodecyl
sulfate (SDS) to denature the proteins. The electrophoresis separation of
proteins performed in polyacrylamide gels because it restrain larger molecules
from migrating as fast as smaller molecules. The protein migration in the
electrophoresis was start from the katode (-) to anode (+). It is because protein
has (-) charge, the SDS give negative charge and denaturate the protein. The
protein migration is based on their mass. After doing several processes, we got
the image of our gel which is the well 1 was loaded with Ep from Tiaras sample,
well 4 was loaded with Es from Tiaras sample, well 5 was loaded with DNA
marker, well 6 was loaded with Ep from Adis sample, and well 8 was loaded with
Es from Adis sample. The length of the gel (L) was 4 cm, and the distance
between the first line/top part and the last line/bottom part (d) of the chosen band
was 2.1 cm. The Ep fraction in the image have to be more thick than the Es,
because it precipitated with ethanol. The thick protein that appears on the gel is
Albumin, which normally have the mass of 67.000 dalton. To measured the
standard curve, the DNA marker have to be visible in order to know the size of
the band. But in our gel result, the DNA marker was not shown. So we had to use
DNA marker from other group (B2-4) that shown the size of the chosen band was
25.000 dalton.
After that, we have to know the data of the axis y = log of size of the band and
the axis x = Rf (Retention factor) which can be got from the formula: d/L (distance
per length).
Then, we made the standard curve for SDS-PAGE based on log of size and Rf
that we got from the previous step. From the standard curve, we got the formula

34

for measuring the molecular mass of the protein in bands in SDS-PAGE, which is
y = -1.2907x + 5.066.
Before we calculate the molecular mass of the protein, we have to know the Rf
(Retention factor) first. The band that we used was the band with the 2.1 cm of
the distance (d = 2.1 cm). So the Rf from that band is :
Rf (x) = d/L = 2.1/4 = 0.525
And in the end, we got the amount of molecular mass of the protein in band
which is 4.381929 (log of size). Because the size is log -1 4.381929, we have to
antilog it and resulting in the different amount of molecular mass of the protein
band is 23,988.33 dalton.
The third experiment that was done is the Enzyme-Linked Immunosorbent Assay
(ELISA) which is done to get the quantitative determination of the antigen AFP
cocentration in human serum. On this experiment, there was an antibody-antigen
binding detection based on enzymatic reaction. AFP (Alpha-fetoprotein) is a
protein that encoded by the AFP gene in human body. AFP has no known
function in healthy adults, so the level of it in healthy adults are low. AFP used as
a biomarker to detect a subset of tumor.
From the experiment that was done by MRIN assistance, the concentration of
antigen AFP in the samples serum are 8.80 ng/ml in sample 1 and 6.25 ng/ml in
sample 2. These results show that both of our samples were healthy or normal
because the results are falls in the normal range (normal = < 20 ng/ml) of healthy
person.
The last experiment that was done in this proteomis session is Colorimetric
determination of blood sugar. Blood sugar is a term used to refer to the level of
glucose in blood. Glucose concentration in blood was effected by diet and
hormones. Glucose is the most important carbohydrate fuel in the body that the
majority of this circulating glucose comes from the diet in the fed state. In this
experiment the blood glucose level was measured from serum sample. We used
the colorimetric in measuring the blood glucose and used the O-toluidine method
to determine or measuring the BSL by forming blue-green complex when it was
boiled (with acetic glacial acid).

35

In the experiment the glucose standard was used to determine the standard
glucose absorbance. The different concentration of glucose standard were used
by each group (our group used 100 mg/dl), so it resulting the variety of standard
absorbance. And the standard curve was made after that.
From the standard curve, we got the formula for measuring the blood sugar level
in the samples. The formula is y = 252.22x + 4.8764. The meaning of the
statement R2= 0,99531 is the linear shape was valid or can be used. To calculate
the blood sugar level in the samples, the absorbance (x) of each sample also
have to be known. The absorbances that we got are Tiaras is 0.2625 and Adis is
0.3625.
In the end we got the result which is the blood sugar level in Tiaras serum is
71.08 mg/dl and the blood sugar level in Adis serum is 87.23 mg/dl.
After doing these whole experiments in the proteomic session it concludes that
the protein concentration of humans blood can be determined by using several
compounds that responsible for the separating process of protein molecule with
other molecules. It also conclude that the molecular mass of protein can be
determined. To know the absolut amount of protein concentration or mass, we
have to be careful in doing the processes. Because an error in process can
makes the result wrong. If the result wrong we can be wrong in examine the
persons condition, because if the result showed the abnormal amount of protein
from persons sample, it indicates to an abnormal health and indicate to diseases
that related to the protein. Whereas, the abnormal amount of the result might be
the result of the human error in doing the processes.

36

REFERENCES
1.

Anonymous. Laboratory Protocol For Fundamental Medical Science

1.Tangerang: Faculty of Medicine-Universitas Pelita Harapan. Mochtar Riady
Institute For Nanotechnology. 2010.

2.

http://www.medterms.com/script/main/art.asp?articlekey=6554

3.

http://themedicalbiochemistrypage.org/protein-structure.html

4.

http://ww2.chemistry.gatech.edu

5.

http://www.ehow.com/about_5502288_blood-plasma-regulations.html

6.

http://www.mcb.uct.ac.za/sdspage.htm

7.

http://en.wikipedia.org/wiki/Protein_precipitation

8.

http://en.wikipedia.org/wiki/Protein_precipitation

9.

http://homepages.gac.edu/~cellab/appds/appd-g.html

37