European University Cyprus

School of Medicine
First Year
Semester 1
Biology lab report
Supervising Professor: A. Stephanou

SDS-Polyacrylamide gel electrophoresis

Students Names:
Andreas Sarantopoulos (Results)
Anna Siebert (Methods and Materials)
Haris Papadopoulos (Abstract)
Ioanna Pappa (Introduction)
Pigi Karavokyri (References)
Polyna Antoniou (Discussion and Conclusion)
Team D4
SDS-PAGE LAB REPORT [Publish Date]

Contents
Abstract……………………………………………………………………page 2
Introduction………………………………………………….……………page 3
Materials and Methods……………………………………………………page 4
Results……………………………………………………………………..page 5
Discussion and Conclusion………………………………...……………..page 6
References……………………………………………………..…………..page 7

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 SDS-PAGE LAB REPORT [Publish Date]

Abstract
The objective of SDS-PAGE experiment is to depict the relationship between molecular mass
and electrophilic mobility for a series of molecular weight standards to determine the purity of
molecular weights between Actin, BSA and Thioredoxin and which of them is the unknown
protein analyzed. This procedure is used to determine protein subunit composition, verify
homogeneity of the protein sample, and purify proteins for use in other applications. The
system consists of two gels – a resolving (running) gel in which proteins are resolved on the
basis of their molecular weights (MWs) and a stacking gel in which proteins are concentrated
prior to entering the resolving gel. From the sample was that was acceptable there were
distinct bands made on the gel at various distances. This indicated that there were proteins of
different weights, the smaller proteins traveled the farthest and also weighed less. Thicker
bands indicated a larger concentration of proteins of similar sizes and weights. In this study,
the unknown protein was found to be the BSA while Y protein had a significant larger
concentration comparing to the Z protein. The mobility of a molecule is also affected by the
buffer system and the strength of the electrophoretic field used for the separation.

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 SDS-PAGE LAB REPORT [Publish Date]

Introduction
SDS-PAGE also known as sodium dodecyl sulfate-polyacrylamide gel electrophoresis is used
for the separation of proteins and nucleic acids (Rockland-inc.com, 2019). In this method, the
proteins are separated according to their molecular weight, where the migration of the proteins
takes place through the gel matrix (Bitesize Bio, 2019). The system contains two gels, where in
the resolving gel the proteins will be separated according to their molecular mass and the
stacking gel, that will concentrate the proteins before entering the resolving gel. In the gel
matrix, the smaller molecules will migrate further compared to the large molecules due to the
limited resistance that exist between the matrix gel and the molecules. The polyacrylamide gel
is primarily used in order to withdraw the large molecules from migrating in the same velocity
as the small molecules and the rate of the migration will depend on the structure and the charge
of the proteins (Rockland-inc.com, 2019). Moreover, Bromophenol blue is used that is an
indicator dye and a migration indicator, where the dye is observed where the proteins run
(Scribd, 2019).

Additionally, in the experiment, the electrophoresis mobility plays a huge role since the
proteins are going to be differentiated according to the length, conformation and the charge
of the molecules (Rockland-inc.com, 2019). An anionic detergent is used in the experiment,
which is known as the SDS. The purpose of the SDS is to dissolve the molecule with a negative
charge and eliminate the influence of the structure and the charge of the protein by linearizing
them (Rockland-inc.com, 2019). The polypeptide chain will bind to the SDS in terms of the
molecular weight, since the SDS is able to destroy the structure of the protein, and thus the
negatively charged molecules will be attracted to the anode in the electric field. Due to the
binding of the SDS with the polypeptide chain, there is going to be an equal distribution of
charge per unit mass (Ruf.rice.edu, 2019).

The general method that was carried out when trying to discover the unknown protein was
labelling the three samples that were available, heating them up at 70C for 10 minutes after
adding the Laemmli buffer. Then, each sample is loaded on the gel, where the process of
electrophoresis takes place and the gel is run and stained for a period of time. Finally, the gel
is destained and the results are obtained, where the identification of the molecules based on
their molecular weight is observed.

Furthermore, the expected results were that there was going to be multiple protein bands on
one of the ladders, which it will indicate that it is a cell extract. The protein with the higher
concentration was expected to have a thicker protein band compared to the protein with the
lower concentration. Further, the unknown protein was expected to be either actin, BSA or
thioredoxin, where the molecular weight of the unknown protein is going to be collected and
compared with the molecular weight of the proteins stated above. Actin, BSA and thioredoxin
have a molecular weight of 42 kD, 66,3 kD, 12 kD respectively.

In conclusion, this report is conducted in order to obtain to analyze the pattern of bands on a
stained SDS-PAGE gel, to estimate the molecular weight of a protein from its migration on
SDS-PAGE gels, to identify the high and low concentration of the unknown protein samples,
and observe the difference of running a cell extract and a single protein.

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 SDS-PAGE LAB REPORT [Publish Date]

Materials and Methods

Materials:

- Electrophoresis chambers
- SDS-Page gels
- Cell extract from MiaPaCl2
- Protein sample
- Protein ladder
- Running buffer
- Staining buffer (10% acetic acid | 25% methanol | 0.05% Coomassie Brilliant Blue G-250)
- Destaining buffer (10% acetic acid | 30% methanol | 60% distilled H2O)
(Rockland-inc.com, 2019)

Methods:

1. Set up the electrophoresis apparatus.

2. Addition of a tank buffer solution to each of the three samples X (unknown protein,
concentration x), Y (also unknown protein, concentration y) and Z (cell extract).
Afterwards heat the samples in a water bath for approximately fifteen minutes at 70C.
3. Load 10μl of each sample on the prepared gel.
4. Run electrophoresis at 120V for one hour until the dye is nearly at the bottom of the gel.
5. Stain the gel with staining buffer on a rocking table for about fifteen minutes.
6. Rinse the gel afterwards two times for ten minutes with a distaining buffer (also on a
rocking table).
7. Leave it in deionized water bath overnight to enlighten.
8. The next day determine the protein by comparing it to the protein ladder.

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 SDS-PAGE LAB REPORT [Publish Date]

Results
For the samples to be observed and analyzed the distaining solution should first be removed
and the deionized water should be added. The result of that procedure can be seen in Figure
1.

Figure 1: The samples X, Y and Z after one night on the rocking board. The first three
columns after the marker constitute the samples of team D4.

The first column on the left side of Figure 1 is the protein ladder, which serves as a measure
of comparison for the identification of the molecular weight of the unknown protein. In addition
to that, in the first sample (Sample X) the migration column consists of multiple protein bands.
This observation indicates that the sample X represents the cell extract. As far as columns 2
and 3 are concerned, it can be concluded that they represent the same protein as only one
protein band has formed, and the migration seems to have the same length. At this point, it is
worth mentioning that the band on sample Y appears to be significantly thicker than the one
on sample Z. This means that there is a higher concentration of the unknown protein in sample
Y than in sample Z.

Figure 2: Polypeptide SDS- PAGE Standards

After comparing the migration pattern of the protein in sample Z with the protein ladder
(Column 1) it was observed that the molecular weight of the unknown protein was 63kD (kilo
Daltons). Based on that data and the Polypeptide Standards in Figure 2 it was concluded that
the unknown protein is Bovine Serum Albumin (BSA), which weighs 66.3 kD.

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 SDS-PAGE LAB REPORT [Publish Date]

Discussion
In this experiment, we used the method of the SDS-PAGE in order to achieve protein
separation. Using this method, you observe the movement of the charged molecules in the
electric field with the support of a medium. The medium in this case was the polyacrylamide
gel, which is synthetic, thermo-stable, transparent, strong and chemically unreactive
(Mypracticalreports.blogspot.com). The pore size of the gel can be determined by the amount
of acrylamide used. During the migration of the charged molecules, the smaller protein will
migrate further due to its molecular weight and due to the less resistance, that exists. The
aspects that affect the migration of the charged molecules are the resistance, the molecular
weight of the protein, the structure and the charge of the protein. Moreover, due to the use of
the sodium dodecyl sulfate with the polyacrylamide gel, the migration of the molecules is not
affected by their charge and the proteins will be separated only base on their polypeptide chain
length (Ruo.mbl.co.jp.).

Furthermore, it was concluded that the unknown protein in the first ladder was BSA since the
6th band in that ladder appeared to be the thickest out of all indicating that the unknown protein
was lying on that band. With the use of the BlueStar Plus Prestrained Protein Marker/
Polypeptide Standards, it was found that the molecular weight of BSA is 66,5 kD and since the
molecular weight of the unknown protein was found to be 63 kD, it was assumed that the
unknown protein is BSA due to the a small difference in the value between their molecular
weights.

BSA is a serum albumin protein derived from cows and it is used in the lab experiments due to
its protein concentration. It also helps with the stabilization of the restriction enzymes during
the digestion of the DNA ((Rockland-inc.com, 2019).

In the first ladder, it was also observed that due to the number of the protein bands, it was
concluded that the sample X was a cell extract. Due to the multiple protein bands found, it is
suggested that the cells have multiple proteins in order to help with the coding of our genes
and the basis of the tissues and their functions. Proteins are the building block of bones,
muscles, cartilage, skin and blood (Cold et al., 2019).

Also, in the second and the third ladder, it was indicated that it is the same protein but with a
different concentration. The sample Y appears to have a higher concertation in comparison
with sample Z due to the difference in the thickness of the band, since it appears that the band
in sample Y is thicker in comparison with the band of sample Z.

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SDS-PAGE LAB REPORT [Publish Date]

References
1. UKEssays.com. (2019). Polyacrylamide Gel Electrophoresis: Protein Separation.
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2. Ruo.mbl.co.jp. (2019). The principle and method of polyacrylamide gel
electrophoresis (SDS-PAGE) | MBL Life Science -JAPAN-. [online] Available at:
https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html [Accessed 2 Dec. 2019].
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serum-albumin.aspx [Accessed 2 Dec. 2019].
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(2019). The Benefits of Protein. [online] WebMD. Available at:
https://www.webmd.com/men/features/benefits-protein#1 [Accessed 2 Dec. 2019].
8. Bitesize Bio. (2019). How SDS-PAGE Works - Bitesize Bio. [online] Available at:
https://bitesizebio.com/580/how-sds-page-works/ [Accessed 5 Dec. 2019].
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10. Scribd. (2019). Laemmli Buffer | Gel Electrophoresis | Polyacrylamide Gel
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