Utilization of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel)

Electrophoresis in Protein Purification

Elfira Jumrah1, Sudding2, Alya Amalia Luqman3, Dwiny Zachrina4, Rifqiani Khairunnisa5, Sri
Hardiyanti6, Virginia Dwi Gladinda7*
1,2,3,4,5,6
Jurusan Kimia, Universitas Negeri Makassar, Makassar, Sulawesi Selatan, Indonesia

*Corresponding Address: virginiadinda18@gmail.com

Received: Month date, year Accepted: Month date, year Online Published: Month date, year

ABSTRACT

A protein can be identified based on each level of its structure. Every protein contains at least
primary, secondary and tertiary structures. Only a few proteins have a quaternary structure as well.
The primary structure consists of a linear chain of amino acids. One of the electric field-mediated
separation methods that is often applied to analyze proteins based on their size is SDS-PAGE
electrophoresis. This technique is based on the assumption that after denaturation, the polypeptide
chains covered in SDS micelles have comparable surface charge densities, so that the resulting
differences in electrical migration are based on their size. SDS-PAGE separates protein molecules
based on their particle size and shape. This gel can be made with varying pore sizes which are
determined based on the total amount of acrylamide compound added (gel concentration). The gel
pore size will become smaller as the gel concentration increases so that only protein molecules that
have a small molecular weight can pass through. This measure of the molecular weight of a protein is
useful in mapping proteins (protein profiling). The gel staining used is bromophenol blue which
functions to color proteins because it binds weakly to proteins. The higher the protein concentration of
a sample being electrophoresed, the resulting band will appear clear and thick. However, if the protein
concentration of a protein is low, the resulting band will appear thin.

Keywords: Electrophoresis, Protein, SDS-PAGE

I. INTRODUCTION
Protein is one of the macronutrients that humans really need, because apart from
being an energy contributor, protein also has an important role in the growth and repair of
cells in the body and helps maintain health conditions. Protein needs during childhood to
adolescence are greater in accordance with the growth and development processes that are
occurring during that period (WHO, 2004). Protein is a food substance that is very important
for the body, because apart from functioning as fuel in the body, it also functions as a
building and regulating substance. Proteins are polymers of amino acids linked by peptide
bonds, protein molecules contain the elements C, H, O and N which fats or carbohydrates do
not have (Winarno, 2008). Proteins are polypeptide structures consisting of one or more long
chains of amino acid residues. They perform a wide variety of organismal functions,
including DNA replication, transporting molecules, catalyzing metabolic reactions, and
providing structural support to cells. A protein can be identified based on each level of its
structure. Every protein contains at least primary, secondary and tertiary structures. Only a

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few proteins have a quaternary structure as well. The primary structure consists of linear
chains of amino acids (Sanvictores & Farci, 2022).
In medical applications such as therapy, diagnostics or other research, protein can be
used but must have very high purity and not contain pyrogens. For this reason, protein
purification is very important. Protein purification is a process used to remove impurities and
improve the quality of the protein produced. Protein purification can be carried out using
polyacrylamide gel electrophoresis which allows protein grouping based on molecular weight
and charge. One method that can be used for protein purification is the SDS-PAGE method.
The SDS-PAGE method is a protein separation based on differences in protein molecular
weight in an electric field (Khatmizarullah, et al, 2021). The mechanism for determining the
molecular weight begins with the immunoglobulin that has been obtained being inserted into
the gel well at the top, where the gel is a collecting gel buffer with larger pores (Senggagau &
Manja, 2020).

II. RESULTS AND DISCUSSION

a. Electrophoresis
Electrophoresis is a technique for separating a particle or species or ion or
colloidal particle based on its ability to move through a conductive medium, usually
a buffer solution, in response to an electric field. Technically, electrophoresis is the
term given to the migration of charged particles as a result of direct electric current
or DC (Direct Current). Gel electrophoresis is one of the main techniques in
molecular biology. The basic principle of this technique is that DNA, RNA, or
proteins can be separated by an electric field. The buffer or buffer solution used in
the electrophoresis process functions to maintain pH balance and provide ions for
conductivity during the electrophoresis process. In this case, the molecules are
separated based on their rate of movement by electromotive forces in the gel matrix.
The transfer rate depends on the molecular size, agarose gel concentration, DNA
fragment size, voltage, concentration and pH of the buffer used, as well as the
concentration and ability of intercalate dyes such as EtBr (ethidium bromide) used in
staining (Siswoyo, 2019).
b. SDS- PAGE (Sodium Dodecyl Sulfate-Gel Polyacrylamida )
One of the electric field-mediated separation methods that is often applied to
analyze proteins based on their size is SDS-PAGE electrophoresis. This technique is
based on the assumption that after denaturation, the polypeptide chains covered in
SDS micelles have comparable surface charge densities, so that the resulting
differences in electrical migration are based on their size. In the reduction mode of
analysis, reducing agents such as mercaptoethanol, dithiothreitol, etc., inhibit the
disulfide bridges of proteins making it possible to analyze their subunits. In fact,
SDS-PAGE is considered an automated instrumental version of the old model
sodium dodecyl sulfate polyacrylamide plate gel electrophoresis (Hajba, et al, 2023).
The use of SDS PAGE electrophoresis is that it can provide an overview or
visualization of the size of the target protein, where there may be other impurity
proteins present. The more protein impurities there are, the visualization shows the
purification quality is less good. Meanwhile, the good quality of the purification
results can be demonstrated by visualizing a single protein band that is thick and has
minimal contaminants (Alami, et al, 2022). This electrophoresis uses two types of
gel, namely separating gel and stacking gel. Separating gel functions to separate
proteins based on molecular weight. Stacking gel functions to equalize the protein

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 starting point before running. The use of SDS whose function is to denature proteins
because SDS is a detergent which causes bonds in proteins to be broken to form
proteins that can be eluted in the gel, SDS to make negatively charged proteins,
mercaptoethanol functions as a base to cut disulfide bonds in proteins so that the
protein structure becomes linear (Oktaviani, et al, 2024). Proteins that are denatured
with SDS will produce negatively charged proteins so that when placed in an electric
field they will move to the positive pole or anode. The migration of proteins in the
gel is proportional to their size so that smaller proteins will move faster than large
proteins. So that proteins that move in an electric field are separated based on their
molecular weight, a porous matrix is needed. The matrix used is polyacrylamide, a
polymer whose monomer is acrylamide (Kembaren & Rachman, 2005).
Proteins have quaternary, tertiary, secondary, and primary structures. In
electrophoresis, the primary structure is needed for protein migration on the
polyacrylamide gel. Heating is needed to denature the protein so that it becomes the
primary structure, but if the heating is at a temperature that is too high for a long time
it will result in total denaturation resulting in damage to the protein which causes the
protein to not be visualized on the polyacrylamide gel. Apart from that, by heating
we can find out and determine the resistance properties of proteins to certain
temperatures.
Proteins will experience changes in chemical structure due to heating or
denaturation, namely breaking bonds in molecules. The development of denatured
protein molecules will reveal the reactive groups in the polypeptide chain. Next,
rebinding will occur to the same or adjacent reactive group. If enough bond units are
formed, the protein will coagulate. If the bonds of the protein reactive groups hold all
the liquid, a gel will form. However, if the liquid separates from the coagulated
protein, the protein will precipitate. In the denaturation process, protein peptide
bonds cannot be completely broken because the primary structure of the protein
remains the same after the denaturation process occurs. Proteins can denature at
temperatures of 50–80°C. The denaturation process in this protein can be caused by
many factors, such as the influence of heating, pH, salt, or stirring. Each method has
a different effect on protein denaturation. Applying heat can have an effect on
protein (Triyono, 2010).
In general, proteins are colorless, so it is necessary to provide a dye that is able
to detect the location of the protein during the electrophoresis process. With color,
the protein can be seen and electrophoresis can be stopped before the protein drops
out of the gel. One example of a dye that can be used, namely bromophenol blue,
functions to color proteins because it can bind weakly to proteins (Silitonga, 2022).
Small proteins will move faster than larger proteins. The relative molecular mass
(Mr) of a protein can be measured using a standard protein (protein marker) whose
Mr is known by comparing the relative mobility value (Rf). Rf protein is a
comparison of the distance between the starting point to the protein band and the
distance from the starting point to the end point of electrophoresis (Febrina, et al,
2022). The SDS-PAGE method has a relatively shorter testing time.
The results of the SDS-PAGE method are in the form of protein bands that
precipitate, after staining the protein bands are read based on molecular weight. The
flow of protein molecules in the gel will form a protein band pattern. Thick protein
bands indicate the number of proteins with the same molecular weight at the same
band position. Bands can form or appear due to a number of sample particles getting

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 stuck at certain points in the gel due to the electric charge on the sample moving
towards the cathode (Hames, 2004). The gel that has been stained and analyzed
shows protein bands forming in each well. If the volume injected into the gel well is
larger, the resulting ribbon will be thicker. This follows the principle of the
movement of charged molecules, which means that the electric field allows charged
molecules to move freely, and molecules with the same charge and size will collect
in the same band or adjacent bands (Salsabila, 2024).
c. SDS (Sodium Dodecyl Sulfate)
SDS is an anionic detergent that can coat proteins, largely in proportion to
their molecular weight, and impart a negative electrical charge to all proteins in the
sample. Glycosylated proteins may not migrate, as it is expected that protein
migration is based on its molecular weight and polypeptide chain mass, rather than
its attached sugars. The use of SDS functions to denature proteins because SDS acts
as a detergent which causes bonds in proteins to be broken to form proteins that can
be eluted in gel (Oktaviani, 2019).
d. Gel Polyacrylamida
Polyacrylamide gel (PAG) is the most common matrix for protein
electrophoresis. Amino, carboxylate, and imidazole groups in proteins are the main
contributors to the net charge of proteins. At extreme pH, groups such as hydroxyl,
guanidino and sulfhydryl can also influence the net charge. The charge contribution
of this group depends on the pKa value and the pH of the surrounding medium. The
net charge of a protein depends on the isoelectric point (pI) and the pH of the
environment. At pH values above pI, the net charge of the protein is negative. In
polyacrylamide gel electrophoresis (PAGE), charged proteins migrate in an electric
field and their mobility depends on the net charge or charge-to-mass ratio. The PAG
matrix also has a filtering effect on protein migration which can help their separation
(Sharma, 2020).
e. Basic Principles
The basic principle of this method is protein denaturation by SDS with
molecular separation based on molecular weight by electrophoresis method using
polyacrylamide gel. When the protein is transferred into a gel and electrophoresed
using electricity, the protein migrates through the pores of the polyacrylamide gel.
Proteins will separate based on molecular size. The level of purity of a protein can
also be seen from the SDS-PAGE electrophorgram in the form of protein bands. The
more protein bands are formed, it shows that the protein is not pure. The
concentration of extract protein affects the quality of the protein bands formed in the
process of analyzing protein characteristics and the speed of protein band formation
(Putri, et al, 2021).
f. Image of SDS-PAGE Results

Figure 1 Zymogram results using casein substrate with sample concentrations of 10 µL, 15 µL and 20 µL. (1: band 1, 2:
band 2, and 3: band 3 (Agustini, 2019)

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 Figure 2 SDS-PAGE and western blot detection of the a0 subunit (Takaiwa, 2019)
g. Equality
The results of electrophoresis in the form of bands can be calculated by
calculating the Rf (Retardation Factor) of each band using the formula:

Then the Rf value is entered into the linear regression equation with the formula:
Y= a + bX
Information:
Y = molecular weight
X = sample Rf value

III.CONCLUSION
SDS electrophoresis is an analytical technique for separating proteins based on their
molecular weight. When proteins are separated by electrophoresis through a gel matrix, the
smaller proteins migrate more quickly due to the less resistance of the gel matrix. Other
influences on the rate of migration through the gel matrix include protein structure and
charge. In addition, the higher the protein concentration of a sample, the clearer the resulting
band will appear. However, if the protein concentration is low, the resulting band appears
thin.

IV. REFERENCES
Alami, E., N., A., Sulistyaningsih, E. & Kusuma, I., F. (2022). Optimasi Purifikasi Protein
Rekombinan CIDRα-PfEMP1 Plasmodium falciparum dengan Kromatografi Afinivtas. Jurnal
Ilmu Dasar. Vol. 23(2).
Febrina, M., Setyahadi, S., & Churiyah. (2022). Purifikasi dan Karakterisasi Enzim Kolagenase dari
Bacillus Sp. KUB BPPT CC dengan Menggunakan Substrat Kolagen dari Kulit Ceker Ayam.
Jurnal Ilmu Farmasi. Vol. 13(1).
Hajba, L., Jeong, S., Chung, S.D, & Guttman, A. (2023). Capillary Gel Electrophoresis of Proteins.
Historical overview and recent advances. https://doi.org/10.1016/j.trac.2023.117024
Hemes, B.D. (1998). Gel Electrophoresis of Proteins. New York: Oxford University Press.
Khatmizarullah, Savalas, L., R., T., & Anwar, Y., A., S. (2021). Isolasi Enzim Lipase dari
Endosperma Kelapa sebagai Bahan Referensi Petunjuk Praktikum Biokimia. Chemistry
Education Practice. Vol.4 (3). ISSN: 2656-3940.
Kembaren, R.F. dan Rachman, E. A. G. 2005. Karakterisasi Protein Rekombinan. Makalah kursus
singkat: Perkembangan terkini dan Prospek Protein Rekombinan dalam Bidang Industri.
School of PharmacY lTB, Bandung. 13-17 Desember 2005. Pages 27-30.

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HAYYAN JOURNAL: Volume xx Number xx, 2024
Oktaviani, E., & Ekaningtias, M. (2019). Analisis Protein Isolat Bakteri Escherichia Coli Bl 21
Menggunakan Sodium Dodecyl Sulphate – Polyacrylamide Gel Elektrophoresis (SDS-
PAGE). Jurnal Pendidikan Biologi dan Sains (PENBIOS). Vol.4(1).
Putri, A., M., Uju, & Hardiningtyas, S., D. (2021). Pengaruh Jenis Pelarut dan Ultrasonikasi Terhadap
Ekstrak Fikoeritrin dari Kappaphycus alvarezii. JPHPI. Vol. 24(2).
Salsabila, Amalia, L., & Nurlaela, R., S. (2024). Perbandingan Profil Protein Daging Ayam dan
Daging Tikus Menggunakan SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis). Jurnal Karimah Tauhid. Vol. 3(5).
Sanvictores, T., & Farci, F. (2022). Biochemistry, Primary Protein Structure. National Library of
Medicine. Senggagau, B., & Bond, M.M. (2020). Aktivitas Imunoglobulin Y Anti-Wssv pada
Serum dan Telur Ayam. Jurnal Biologi dan Pembelajarannya. Vol. 15(1).
Sharma, N., dkk. (2020). Separation Methods for Milk Proteins on Polyacrylamide Gel
Electrophoresis: Critical Analysis and Options for Better Resolution. Journal Pre-proof.
Elsevier. https://doi.org/10.1016/j.idairyj.2020.104920.
Silitonga, M., Chandra, D., Fitri, R., & Nanda, F. (2022). Analisis Faktor Yang Mempengaruhi
Pertumbuhan Kultur Sel CHO (Chinase Hamster Ovarium) Pada Pembuatan Obat
Trastuzumab Di Pt. Bio Farma. Jurnal Farmanesia. Vol. 9(1). HAYYAN JOURNAL: Volume
xx Number xx, 2024
Siswoyo, A., B. (2019). Ekstraksi DNA. Jurnal Budidaya Perairan. Vol. 4(5).
Triyono, A. (2010). Mempelajari Pengaruh Penambahan Beberapa Asam pada Proses Isolasi Protein
terhadap Tepung Protein Isolat Kacang Hijau (Phaseolus radiatus L.). Seminar Rekayasa
Kimia dan Proses. ISSN 1411 4216.
Winarno, F. G. (2008). Kimia Pangan dan Gizi. Jakarta: PT Gramedia Pustaka Utama.
World Health Organization [WHO]. (2004). Energy and protein requirements. Geneva: FAO/
WHO/UNU.

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