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Group No.

4 Date Submitted: September 8, 2016


Date Performed: August 30, 2016

Exercise No. 2
Analysis of Human Blood Proteins Using SDS-PAGE

Abstract

Polyacrylamide gel electrophoresis (PAGE) with the use of sodium dodecyl sulfate
(SDS) is an efficient way of determining the molecular weights of proteins in a given sample
by means of separating such proteins that constitute it. This process takes advantage of the
charges in the PAGE-SDS, in which SDS denatures the proteins in the sample, resulting in
these proteins traveling towards the positive front solely based on their molecular weights.
Human blood plasma and red blood lysates were used in this experiment to identify the
molecular weights of the proteins that constitute the samples. Results showed that
hemoglobin (found in red blood lysates) weighed less than serum albumin (similarly found in
red blood lysates), with hemoglobin weighing only 15 kDa as opposed to serum albumins 75
kDa.

Introduction

The cells in the blood all come from the bone marrow. They start as stem cells and
mature into three main types of cells red blood cells, white blood cells, and platelets. Red
blood cells (or erythrocytes) are the most common type of cell in the blood. In humans, the
RBC lacks nuclei which allows for more room to store hemoglobin, the oxygen-binding
protein, that helps the RBC transport oxygen. It is also the densest component of the blood.
When left isolated, the blood usually separates into three layers, one of which is the RBC.
The other layer is that which composes the WBC and the platelets. WBC circulates in the
blood to aid in the immune response of the human body. The least dense layer, which was
found on the uppermost part, is the plasma. Plasma is mainly water and it forms about 60%
of the blood. Together with water, it also contains proteins such as albumin, antibodies, and
enzymes; sugars; and fat particles (Bethesda 2005).

Electrophoresis is one of the techniques in Biology used to separate molecules of a


given sample. It relies on the difference in electric potential to migrate different proteins
according to their own shape and size in an electric field. Separation of protein, specifically,
is done using polyacrylamide gel electrophoresis with the presence of sodium dodecyl
sulfate. Proteins are driven by applied current in a gelled solution. SDS, a negatively
charged solution, binds in large numbers to all protein molecules which causes the proteins
to unfold. SDS-PAGE can be used to determine the molecular mass of different kinds of
proteins by comparing positions of bands to the molecular marker. This was done in the
blood protein and plasma protein samples used in the experiment (Karp 2010).

In this experiment the human blood proteins were extracted into two components
the RBC proteins and the plasma proteins. These proteins were subjected to SDS-PAGE
and their molecular masses were obtained through a comparison group, which is the
molecular weight marker.

Methodology

Deslate, Manalang, Salazar, Santos


The experiment started with the preparation of the SDS-PAGE. First, the gel
apparatus (Bio-Rad Mini Protean Tetra Cell) was set up. After which, the running gel
monomer was prepared. It consisted of monomer solution, running gel buffer, 10% SDS,
ddH2O, 10% APS, and TEMED. When everything was combined, the running gel was
poured to the gel apparatus. It was overlaid with n-butanol for the gel to polymerize, then it
was isolated for 20 minutes.

While waiting for the running gel to polymerize, the volunteer was asked to sign a
consent. Then, the hands of the volunteer were cleaned with soap and 70% rubbing alcohol.
With a sterile lancet, the skin of the fingertip was pierced and a micropipettor was used to
collect 20-25 L blood from the pierced fingertip. This was transferred to a 1.0 mL
microcentrifuge tube with 40 L of 0.10 M NaCl. In order to mix thoroughly, the
microcentrifuge was tapped repeatedly. The sample was, then, centrifuged for two minutes
at 6,500 rpm to distinguish the pellets of red blood cells from the plasma. The serum was
transferred into another 1.0 mL microcentrifuge. This was set aside in the freezer before
resuspending the red blood cells (pellets) in 40 L of 0.065 M KCl. The sample was vortexed
vigorously and then centrifugation was done for five minutes at maximum rate which is
14,500 rpm. After centrifugation, the clear red supernatant was transferred into a new 1.0 mL
microcentrifuge tube which was then set aside in the freezer.

As the running gel polymerized, the n-butanol was decanted and the gel was washed
with running gel overlay. Then, the stacking gel was prepared and poured to the gel
apparatus. This consisted of monomer solution, stacking gel buffer, 10% SDS, ddH2O, 10%
APS, and TEMED. The comb was then inserted and the gel was allowed to polymerize
completely for another 20 minutes.

The last part of the experiment was the electrophoresis. The samples were diluted
2:1 with the sample treatment buffer in a 1.5 mL microcentrifuge. Prior to loading, the
samples were heated at 95 oC for four minutes. 30 L of each sample was then loaded
together with 5 L of protein molecular weight marker. After that, the tank was filled with tank
buffer. Running of the gel was then performed at a constant 200 V for 30-40 minutes. The
gel was removed from glass plates and then transferred to a staining pan with the
Coomassie Brilliant Blue staining solution. This gel was stained for four hours, lasting
overnight, followed by the transferring of the gel to the destaining solution. It was destained
until the gel was almost transparent, and then stored in distilled H2O. The results were
documented afterwards.

Results and Discussion

The experiment made use of gel electrophoresis, a separation and analysis


technique used to examine macromolecules (e.g. proteins) and their fragments. In the
process of polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS),
an anionic detergent, is used in protein samples to linearize proteins and to impart a
negative charge to linearized proteins.
A chemical buffer was used (1.5 M Tris-Cl) to stabilize the pH value to the desired
value within the gel itself and in the electrophoresis buffer. This particular buffer was used,
because the choice of buffer affects the electrophoretic mobility of the buffer counterions and
thereby the resolution of the gel. Acrylamide was used to greatly accelerate polymerization.
When dissolved in water, slow, spontaneous autopolymerization takes place to form long
single-chain polymers. A solution of these polymer chains does not form a gel, because the
chains slide over one another, hence the need to use a stacking gel. A staining solution
was also used, because proteins and nucleic acids are mostly colorlesstheir progress

Deslate, Manalang, Salazar, Santos


through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known
electrophoretic mobility are therefore usually included in the PAGE sample buffer. A staining
solution containing CBB (Coomassie Brilliant Blue R) was used as the protein stain in this
solution.

Figure 1. SDS-PAGE of blood samples.

Distance of gel front: 1000mm


Molecular Weight Log MW Distance Travelled Rf
(kDa) (with respect to
front) in mm

225 2.35 10 0.01

150 2.18 50 0.05

75 1.88 350 0.35

50 1.70 470 0.47

35 1.54 600 0.6

25 1.40 750 0.75

15 1.00 950 0.95


Table 1. Molecular weight and Rf values of the sample.

Deslate, Manalang, Salazar, Santos


Figure 2. Graph of molecular weight and Rf with trendline equation and correlation
coefficient.

The major components of blood include the following: (1) Plasmawhich contains
the major protein, albumin (2) red blood cells--contains the protein, hemoglobin, (3) white
blood cells, and (4) platelets. The major function of hemoglobin is to transport oxygen from
the lungs to the body's tissues and then transport carbon dioxide out of the tissue back to
the lungs. One hemoglobin molecule in a mammal can carry up to four oxygen molecules.
Hemoglobin also carries nitric oxide, an important regulatory molecule, and releases nitric
oxide when it releases oxygen. Hemoglobin is a complex protein molecule made up of four
subunits of polypeptides, or globins, which are chains of amino acids. These globin subunits
bind to non-protein heme groups containing an iron ion, which can bind to oxygen. These
iron ions are in turn enclosed in rings called porphyrins. Albumin, the major protein of
serum, can be detected in the SDS-PAGE.
Seven protein bands were found in the SDS-PAGE, while three protein samples were
detected, particularly albumin,hemoglobin alpha and beta subunits, and microglobulin. The
bands seen in the gel indicate the molecular weights of each protein components such that
the farther a band has traveled, the lighter its molecular weight is. Based on the
electrophoresis gel, hemoglobin weighs lighter than albumin, with albumins estimated
molecular weight being 75 kDa and hemoglobin alpha and beta subunits' being 15 kDa.
Note, however, that these are merely approximations based on the acquired results because
the sample bands are not properly aligned with the kDa band reference.

The preparation for the polymerization of the gel needs to be be properly executed so
that proper polymerization commences. Acrylamid is C3H5NO and forms linear polymers in
the polymerization process. Bis-acrylamide, on the other hand, is C7H10N2O2 and cross
links the existing linear polymers. Thus not adding these would result in improper
polymerization of the gel, making it difficult for the samples to travel through the gel later in
the experiment. Polyacrylamide is (C3H5NO)n.The stacking gel has large sized pores that

Deslate, Manalang, Salazar, Santos


allow the proteins to migrate freely and get stacked at the interface between stacking gel and
running gel (hence the name stacking gel). The purpose of this is to make sure that the
proteins start migrating from the same level, since they are separated based on their mass
(the effect of their charge is overcome by SDSs high negativity). It is also important to note
that the gel should be mixed gently during preparation because excessive aeration would
interfere with the polymerization process of the gel. On the other hand, the running gel
allows the separation of the proteins in the sample based on their molecular weight. In the
process of polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS), an
anionic detergent, is used in protein samples to linearize proteins and to impart a negative
charge to linearized proteins.

In the case that bubbles formed while creating the polymerized gel, the protein will
not move correctly through the gel during electrophoresis. The gel system should provide a
constant electric field for the protein to migrate through. If there are air bubbles in the gel, the
electric field will not be constant as the electric current inside and outside the air bubbles will
vary. If the gel solution is exposed to air for a long period of time, there is a possibility that
the rate of polymerization will slightly decrease. The same thing also happens occurs if the
APS contained any contaminants.

Applications of PAGE include paternity tests and peptide mapping. In a paternity test,
DNA samples of the child, the mother, and the alleged fathers are usually subjected to SDS-
PAGE. In the result, one of the bands in the childs sample should match (i.e., should have
traveled at approximately the same distance) as at least one band from each parent. Thus,
the alleged father with the most number of matching bands is most likely the biological
father. Peptide mapping, on the other hand, is used to identify modifications in amino acid
sequences (Simpson 2003); such that when a normal amino acid sequence and a modified
amino acid sequence is subjected to SDS-PAGE, differences in band traveled distances
between the two samples may indicate modifications of specific amino acids.

Conclusions and Recommendations

The data generated from the exercise show comparative molecular weights of
proteins found in the blood, specifically in red blood cell (RBC) lysates. Three major proteins
found in the blood, hemoglobin, microglobulin, and serum albumin, have significantly
different molecular weights based on the SDS-PAGE analysis of human blood. One protein
travelled a longer distance from the gel front, with a calculated relative mobility (Rf) value of
0.95. This has the smallest molecular weight at 15kDa. Based on the theoretical molecular
weights of the proteins found in RBC, this are the hemoglobin alpha and beta subunits. The
other protein has a calculated Rf value of 0.75, with a molecular weight falling somewhere
between 15-25kDa, but nearer to 25kDa based on the SDS-PAGE analysis. This is
microglobulin, which weighs 25kDa. Lastly, another protein was detected using SDS-PAGE,
with the least mobility at 0.35. This is serum albumin, which weighs 75kDa. To encapsulate,
among the proteins detected in the exercise, hemoglobin is the lightest, followed by
microglobulin, and serum albumin has the biggest molecular weight.
To further improve the exercise, crucial procedures should be executed carefully and
the state of the sample should be keenly monitored. For instance, prior to electrophoresis,
the RBC pellets were vortexed and centrifuged until it was a clear red supernatant. However,
in some cases, it may not immediately become clear red, and centrifugation may still be
necessary. This may affect the electrophoresis process per se if the sample was not properly
vortexed and centrifuged. Also, after electrophoresis, the gel should be transferred to the

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staining pan with CBB very cautiously. This is because the gel is fragile, thus susceptible to
damages if not carefully handled. If mishandled, some parts of the gel may be torn, causing
a significant problem in interpreting the distances travelled by the proteins from the sample.
Overall, the exercise can be improved through knowing the different stages into which the
sample will transform before the process of electrophoresis and by carefully handling the
SDS-PAGE gel.

References

Bethesda, DL. 2005. Chapter 1, Blood and the cells it contains. In Blood Groups and Red
Cell Antigens. United States: National Center for Biotechnology Information.

Constantino, MK. 2015. Exercise 2: Analysis of Human Blood Proteins Using


SDS-PAGE. In Cell & Molecular Biology Laboratory Exercises. Quezon City: Ateneo
de Manila University Department of Biology.

Karp G. 2010. Cell Biology. Singapore: John Wiley & Sons.

Simpson, RJ. 2003. SDS-PAGE Peptide Mapping Procedure [Internet]. [cited 8 September
2016]. Available from: http://cshprotocols.cshlp.org/content/2007/2/pdb.prot4591
.abstract

Deslate, Manalang, Salazar, Santos