BIOANALYTICAL CHEMISTRY LABORATORY BSB2442

EXPERIMENT 5:
ELECTROPHORESIS

Group Morning session (Section B): 9am – 10am

Group member 1. Nurul Syafiqah Binti Nazari
(SB20039)
2. Chen Yoke Ching
(SB20041)
Course BSB2442 Bioanalytical Chemistry
Laboratory
Section B1
Date of assignment 13 December 2021
Date of assignment submission 3 January 2022

Report Outline Person-In-Charge

Introduction Nurul Syafiqah Binti Nazari

Methodology Nurul Syafiqah Binti Nazari

Results Chen Yoke Ching

Discussion Chen Yoke Ching

Conclusion Chen Yoke Ching

References Nurul Syafiqah Binti Nazari

Appendices Chen Yoke Ching

INTRODUCTION

Electrophoresis is a technique used in laboratories to segregate DNA, RNA, and protein

molecules depending on its size and electrical charge. To separate molecules, an electric
current is used to move them over a gel. The gel's pores act as a filter, allowing smaller
materials to diffuse more quickly than bigger molecules. The electrophoresis settings can be
changed to separate molecules in a certain size range.

In electrophoresis, charged molecules travel towards the positively or negatively pole

based on their charges. Nucleic acids have a continuous negative charge provided by its
phosphate backbone and move towards to the anode, unlike proteins, which can have either a
net positive or net negative charge. An ion in such an electric field will be exposed to a force.
Depending on the magnitude of the protein's charge, the force will lead it to speed towards to
the cathode or the anode. In fact, there are additional forces at work when ions travel in an
electric field, such as friction. Electrophoresis takes advantage of the fact that various
mobility in an electric field can be separated in this method. Proteins and nucleic acids are
separated by electrophoresis in a matrix, often known as a gel. The gel is usually cast in the
shape of a thin slab with wells for loading the material. The gel is submerged in an
electrophoresis buffer, which contains ions to conduct a current and a buffer to keep the pH
stable. The gel itself is made of either agarose or polyacrylamide, both of which have
properties that are ideal for specific activities.
 Electrophoresis is influenced by a variety of factors. As the molecules go by one pole
to the next, they have cross through into the gel. Larger molecules have a harder time
weaving in and out of the gel matrix. As a general rule, molecules travel faster if they have a
higher net charge, a spherical shape, and a smaller diameter. Electrophoresis is also
influenced by a number of additional parameters, including molecular mass, buffer, and
electrophoretic media, such as paper or gel.

Ions in the buffer convey the charge required for separating in electrophoresis that
separates on the basis of charge. The buffer also regulates the pH inside a restricted range by
providing a reservoir of weak acid and base. This is critical because considerable pH
fluctuations will affect the structure and charge of a protein or nucleic acid, preventing
effective separation. Various buffers are best for keeping the electrophoresis gel at various
pH levels. Acetic acid, boric acid, phosphoric acid, citric acid, glycine, and taurine are some
of the most common buffers utilized by chemists for this objective. The pKa value (acid
dissociation constant) should be close to the required pH in most cases. In order to avoid
conducting too much current, it is recommended to use buffers with a low charge magnitude.

The significance of electricity in biological processes is equally as essential as its

position in technology, and it's put to scientific use in a variety of subtle and surprising ways.
Electrophoresis, the use of an electrical current to modify protein molecules for a variety of
biological study, diagnostics, and production reasons, is one of the most commonly used
techniques in biochemistry. Researchers can generate circumstances that efficiently separate
biomolecules so they can be separated and analyzed by adjusting the electrical current and the
friction produced by the test medium. It also allows researchers to distinguish distinct
molecules by measuring how much they are affected by the current. It's a versatile instrument
with a variety of experimental and biological applications, but there are a handful that stand
out. The detection and study of DNA and DNA fragments is a common application of
electrophoresis. DNA is famous for its consistent negative charge, which means that an
electrical current exerts about identical force on all parts of the molecule. In the testing of
antibiotics, electrophoresis is used in a variety of ways. Testing the purity of an antibiotic is
one of the most common. Electrophoresis, like antibiotics, is helpful in the development and
manufacture of vaccines. A vaccine's goal is to help the body produce antibodies against a
potentially deadly infection, and electrophoresis is a good way to identify such antibodies.
 In the laboratory, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) is often used to separate proteins depending on the molecular weight. It's one
of those strategies that is widely utilized but rarely properly comprehended. SDS is a
detergent that is contained in the SDS-PAGE sample buffer and disturbs the tertiary structure
of proteins when combined with boiling and a reducing agent (usually DTT or beta-ME to
break down protein–protein disulfide links). Folded proteins are reduced to linear molecules
as a result of this process. SDS also coats the protein in a uniform negative charge, which
hides the R-groups' intrinsic charges. SDS is also included in the gel to ensure that the
proteins are linearized and their charges are hidden for the period of the test. The molecular
radius of an SDS-coated protein is the most important element in influencing its mobility.
There will be no charge-based differential migration since the SDS-coated proteins have the
same charge-to-mass ratio. The gel matrix used for SDS-PAGE is polyacrylamide, which is
an excellent choice since it is chemically inert and, more importantly, can be easily built up at
a number of concentrations to provide varied pore sizes, allowing you to modify the
separation conditions to suit your needs.
OBJECTIVES

1. To learn how to prepare stacking and separating gels

2. To understand the principles of electrophoresis
3. To provide a reliable approach for determining substances
4. To learn the purposes of electrophoresis
5. To understand how the separation principle works using the SDS-PAGE
electrophoresis method.

METHODOLOGY

Materials:

1. Glass plates (tape with masking tape)

2. Plate holder
3. Gel tank
4. Lid of gel tank
5. Positive and negative wire
6. Powerpack/power supply
Methods:

Part I: Preparation of a Polyacrylamide Gel (Stacking and Separating Gels)

1. The gel plates, spacers and stand were cleaned as per manufacturer’s instruction.
2. The gel plate was placed on top of the spacer.
3. The gel plate and spacer were slide into the casting frame. The short place was
ensured to face the front.
4. The pressure clamps were locked to lock the glass plates in the position.
5. The spacers and gel plates were assembly on the gel stand for casting the gel. The gel
plate and spacer were ensured to align at the bottom.
6. The casting frame was secured in the casting stand by engaging the spring loading
lever.
7. The gel premix solution was added swiftly using 1 ml micropipette until it is 2.5-3.0
cm from the top of the gel plate without introducing any air bubbles.
8. The comb was placed between the gel plate and spacer carefully.
9. The gel is then ready to undergo electrophoresis.
Part 2: Running and Staining Electrophoresis Gel

1. The gel plates and the spacers with prepared gel were clamp into the casting frame
with the pressure clamps. The gel plates were ensured facing front.
2. The spacers and gel plates were assembled and ensure they are aligned at the bottom.
3. The spacers and gel plates with prepared gel with casting frame were placed vertically
into the electrophoresis tank.
4. The electrophoresis tank was filled with 1X running buffer.
5. The positive and negative wire were connected between the lid of the electrophoresis
tank and the power supply.
6. The gel was run at 120 Volts for 45 minutes.
7. The gel was disassembly carefully and the 1X buffer solution was poured into a new
beaker.
8. The available protein bands on the gel were identified by comparing to the protein
marker.
RESULT

Figure 1 : The result of SDS-PAGE different samples with markers

Type of Sample Number of Visible Bands Estimated Size (kDa)
Non-fat dry milk

= 34
= 29

= 19

= 12
=4
Bovine Colostrum =220

= 88
= 76
= 59

= 35
= 33
= 27

= 17

= 12
=9
Immunoglobin G

= 57

= 28

= 11
=3
Whey = 221

= 24

= 15

=3
Casein

= 71

= 32
= 30
= 28

=4
Bovine Serum Albumin = 220

= 63

=2
Lactoferrin

= 82

=1
Ovalbumin

= 41
= 39

=2
Egg White

= 79

= 50
= 41

=4 = 13
Egg Yolk = 221
= 157
= 94

= 70
= 69
= 60
= 50
= 38
= 35
= 30

= 11 =8
Whole Egg
= 158
= 94
= 78

= 37
= 35

= 13

=7 =9
Zein

= 25
= 23

=2
Soy Flour

= 85
= 70
= 65
= 60
= 43
= 37
= 34
= 32
= 29
= 27
= 24
= 22
= 19
= 16

= 14
Wheat Gluten

= 94
= 88
= 82

= 43
= 38
= 35
= 33
= 31

=8
DISCUSSION

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a

separating method for protein by electrophoresis based on their molecular weight. The
polyacrylamide gel used in SDS-PAGE is inert and does not interact with the proteins.
Polyacrylamide gel forms a matrix. The pore size of the matrix is depended on the
concentration of the polyacrylamide gel. The denaturing buffer which is sodium dodecyl
sulfate (SDS) is an anionic detergent. SDS converts the secondary structure and tertiary
structure of protein into primary structure. SDS break down the hydrophobic interactions and
hydrogen bonds in the protein to produce a linear polypeptide chain. Then, the hydrocarbon
tail of SDS which is polar binds to the side chain of the protein making the protein to become
negatively charged. Therefore, the result of SDS-PAGE is based on the molecular weight of
the protein but not their shape and charge.

An electrical field is applied. The charge from the starting point is negative, and the
frontline is positive charge. The negatively charged proteins will be repelled at the starting
point which is also negative and attracted to the positive charge. The further the protein
travelled, the lower the molecular weight it has. Larger protein has higher molecular weight,
they cannot migrate further because of their molecular weight and they are stuck at the matrix
of the polyacrylamide gel. The bands were stained with Coomassie brilliant blue so that the
bands are visible in blue colour against a white background.

From the result obtained, the band shown at the left side of the Figure 1 which
labelled the molecular size is from a standard of protein. It is used to compare the molecular
size of the sample bands with the molecular size of the standard. The number of visible bands
for the type of sample can be arranged ascendingly, which is Lactoferrin (1), Ovalbumin (2),
Bovine Serum Albumin (2), Zein (2), Immunoglobin G (3), Whey (3), Non-fat Dry Milk (4),
Casein (4), Egg White (4), Whole Egg (7), Wheat Gluten (8), Bovine Colostrum (9), Egg
Yolk (11) and Soy Flour (14).

The number of visible bands in Lactoferrin is the lowest, which is 1. The band shows
the size of polypeptide chain is approximately in 82kDa. The single band in Lactoferrin
indicates there is only one type of polypeptide chain was detected for the protein Lactoferrin.
The number of visible bands for Soy Flour is the highest, which is 14. There are 14 types of
fragments of Soy Flour protein were detected. The size of each type of polypeptide chain are
estimated at 16kDa, 19kDa, 22kDa, 24kDa, 27kDa, 29kDa, 32kDa, 34kDa, 37kDa, 43kDa,
60kDa, 65kDa, 70kDa and 85kDa. There are 14 types fragments of Soy Flour protein were
detected.

The further the protein travelled, the lighter its molecular weight, the shorter the
length of polypeptide chain. Longer polypeptide chain, larger molecular weight, thus it
cannot migrate further because it is stuck in the matrix of SDS gel. The thickness of the bands
indicates number of the type of polypeptide chain present. The thinner the band indicates the
number of the certain type polypeptide chain is lesser. The thicker the band indicates the
number of the certain polypeptide chain is higher.

CONCLUSION

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is an

analytical technique to separate proteins based on their molecular weight by an electric field
applied. The smaller the protein fragment, the shorter the polypeptide chain, the lesser
resistance for them to travelled further in the gel matrix, thus the longer distance they
travelled. The larger the size of protein fragment, the longer the polypeptide chain, the shorter
the distance they travelled in the gel matrix because of higher resistance to the gel matrix.
From the result obtained, Lactoferrin shows one band, which estimated size of the
polypeptide chain is 82kDa. Soy flour shows the highest number of bands which is 14 bands.
The estimated size of the polypeptide chains is 16kDa, 19kDa, 22kDa, 24kDa, 27kDa,
29kDa, 32kDa, 34kDa, 37kDa, 43kDa, 60kDa, 65kDa, 70kDa and 85kDa.
REFERENCES

1. Austin, C. P. (n.d.). Electrophoresis. Retrieved from National Human Genome

Research Institute : https://www.genome.gov/genetics-glossary/Electrophoresis

2. Collins, P. (21 May, 2018). The Purpose of the Buffer in Electrophoresis. Retrieved
from Sciencing: https://sciencing.com/sources-of-error-in-gel-electrophoresis-
12359700.html

3. Decker, F. (13 March, 2018). List of the Applications of Electrophoresis. Retrieved

from Sciencing: https://sciencing.com/purpose-buffer-electrophoresis-6613320.html

4. Dephoff, J. (n.d.). The Purpose of Electrophoresis. Retrieved from Sciencing:

https://sciencing.com/purpose-electrophoresis-5426937.html

5. Oswald, D. N. (11 August, 2021). How SDS-PAGE Works. Retrieved from

bitesizebio: https://bitesizebio.com/580/how-sds-page-works/

6. Pharmatutor. (15 October, 2016). Explain Electrophoresis, its principle and factors
governing it. Retrieved from Pharmacy Infopedia:
https://www.pharmatutor.org/pharma-analysis/explain-electrophoresis-its-principle-
and-factors-governing-it
APPENDICES

Diagram 1 shows the preparation of the gel electrophoresis where the gel premix solution
transferred in the space between gel plates and spacers, then the comb was put between the
gel plates and spacers.

Diagram 2 and 3 show the process of running

electrophoresis gel by connecting the cathode and anode of the power supply to the
electrophoresis tank.