NNNB6013

TEKNIK MAKMAL DALAM SAINS

BIOPERUBATAN

Name: Nur Athirah Binti Hamzah

Matric No: P95599
Tittle: SDS PAGE and Western Blot of Beta-Actin Protein from
Jurkat Cell (Human Brain Endothelial Cell)
Lecturer: Dr. Nurul Farhana Binti Jufri
Date: 24/12/2018
INTRODUCTION:

The types of gel electrophoresis for proteins depending on type of sample to be separated.
In this experiment, SDS-PAGE electrophoresis is carried out to separated protein sample. The
objective of this lab is to determine separate protein of actin from Jurkat cell, which is
accomplished by performing an SDS-PAGE, and also to perform a Western Transfer in the
following lab. Both SDS PAGE and Western Blot are based on charge or pI (isoelectric point)
separation. Western Blot is a type of confirmation test of expression level of a protein using an
antibody and has stronger validity than SDS page. In real life, it is often used as a follow-up test
to confirm the presence of an antibody and to help diagnose a condition.

Beta-actins are made as the target protein from Jurkat cell. This is because it is one of the
most highly-conserved proteins and has molecular weight of 42 kDa. Actins are comprised of three
main isoform groups which are alpha, beta and gamma. The alpha actins are found in muscle
tissues and are a major component of the contractile apparatus. The beta and gamma actins are
present in most cell types as parts of the cytoskeleton and are mediators of cell trafficking,
structural integrity and cell motility. Beta-actin specifically is a cytoplasmic actin that able to give
general expression on all type of cell.

Jurkat cells are immortalized T lymphocytes first derived from the peripheral blood of a
child suffering from leukemia. Originally called JM cells, Jurkat cells found its significance due
to its robust ability to produce interleukin-2. It play importanat role in understanding T-cell
receptor signaling and T-cell activation. Jurkat widely used for studies on protein expression, viral
interactions, and cancer biochemistry, among others.
 During this lab, a Mini-PROTEAN tetra system is set up, and then an SDS-PAGE resolving
gel and stacking gel are prepared. The gel that are prepared to carry out gel electrophoresis for the
protein separation are resolving gel and stacking gel. Initially, the gel cast set is set up and the
leaking test is done to prevent the gel from leaking in the following step. The resolving gel is
prepared initially with 12% concentration using distilled water, 1.5M Tris Hydrochloric Acid (pH
8.8), 10% SDS, 30% Acrylamide, 10%APS and TEMED. The TEMED is put the last as it can
catalyst polymerization and cause the gel to solidifies rapidly. Few drop of water is added on the
surface of the gel to give uniformity to the gel surface. Next, the resolving gel with 5%
concentration is then prepared using 0.5M Tris Hydrochloric Acid (pH 6.8), 10% SDS, 30%
Acrylamide, 10%APS and TEMED. Comb of 10 well is assembled inside of stacking gel and is
let to solidifies before being transferred into electrophoresis tank. These gels are added to the Mini-
PROTEAN tetra system so that they polymerize in the desired conformation with wells on the top
end.

Diagram 1: Mini-PROTEAN tetra system set up for SDS-PAGE process

The Jurkat cell was prepared using 5x loading buffer and heated at 95𝑜𝑜 𝐶𝐶 for 5 minutes.
Sample preparation is crucial step to obtaining accurate separation of the proteins on the basis of
molecular weight depending on type of antigen whether it is extracellular, cytoplasmic, or
membrane-associated. Soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers
that contain the non-ionic detergent. Samples mixed with loading buffer are carefully pipetted into
two wells alternately along with the markers in a well, taking up a total of three lanes. The 10ul
protein marker is loaded at the most last side followed by loading of Jurkat cell with 20ul
alternately into two well subsequently. The electrophoresis was run with voltage power at 100V
for 10 minutes and later by 150V for an hour. The gel is then immersed in Coomasie Blue staining
solution on shaker after the electrophoresis is done. The bands on gel is observed on a light box to
enable clear vision is obtained.

Diagram 2: Denaturation of target protein by SDS reagent from tertiary and secondry
structure into a linear and simper primary structure

This experiment use SDS protein separation and staining with Coomassie Blue stain to
detect protein only at expression level without specific detection. This is a type of denaturing
method as it treats the proteins with anionic SDS detergent (sodiumdodcylsulfate). During PAGE,
the rate of migration of SDS-treated proteins is effectively determined by molecular weight.
Therefore the following separation happens solely by the size of the polypeptide chains in the
polyacrylamide gel.

This was then further tested with Western Blot for the interest protein using polyclonal
rabbit primary antibodies and secondary monoclonal goat antibodies with fluorescence tagged to
be determined using after washing alternately using TBST. This is an immunoassay test method
that detects specific proteins in blood or tissue by combining electrophoresis step and transferring
(blots) the separated proteins onto a nitrocellulose membrane.
 Immunoblotting occurs in six stages which are extraction of protein samples and followed
by resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel
electrophoresis (SDS-PAGE). Multiple protein samples and a control are placed at the end of the
gel in separate lanes. An electrical current is applied to the protein mixture by gel electrophoresis
in a carrier matrix which is SDS-PAGE electrophoresis to move protein samples across the gel and
separate the proteins by size or charge of different individual protein bands by forming bands that
resemble the steps of a ladder. Following the separation of the protein mix the polypeptide bands
is transferring the separated protein bands to a carrier membrane which is PVDF that is put contact
with the gel. This method is called as sandwich which is then transferred to an electrophoresis
chamber. This was then followed by blocking of nonspecific binding sites on the membrane was
done. Later, antibodies was added and followed by detection. The blotted bands are now available
to be treated further for detection of specific proteins with specific labelled or tagged antibody.
Antibodies are used to detect target proteins on the western blot . The antibodies are conjugated
with fluorescent or radioactive labels or enzymes that give a subsequent reaction with an applied
reagent, leading to a coloring or emission of light, enabling detection.

Analysis for identification of a protein of interest in a Western blot is then carried out using
imaging systems by visualizing on the Gel Doc (e.g. luminescence, color reaction,
autoradiography).To this end, the electrophoretically separated proteins in the sample are
compared with a molecular weight standard of a known composition.
 PROTOCOLS

Clean Glass plate+ Spacer

Wipe Glass plate + Spacer plate with 70%

Apply vaseline on side of glass

Attach Glass plate+ Spacer plate together

Put the attached plates into plate holder, clamp, assemble into plate holder

Set up glass plate and spacer again Do leaking test

Pour stacking gel
Pour resolving gel in between Insert comb onto resolving gel
the glass plates (1.5~2.0 cm into (2.0 cm from the
from the end of short plate) Let gel solidified stacking gel end of short plate)

Assemble the plates into the electrophoresis tank

Pour the 1x EB buffer into electrophoresis tank

Take out comb from stacking gel Mix 5x loading buffer+

Protein molecular Jurkat cell
Load 10ul Marker into last side
weight marker (1:1 mix
Heat to 95oC, 5 mins
with 1x loading buffer)
Load 20 ul Sample(Jurkat Cell) into 2 well and store in −200 C
alternately following the well containing marker

Run electrophoresis at 100V for 10

minutes followed by 150V for 1 hour

Take out the gel plates and

Western Blot
remove the stacking gel

Wet resolving gel with buffer

Immerse resolving gel into Coomassie Blue

staining solution on a rocking shaker (30

Wash the gel with water (a few minutes)

Place the gel on a light box

 Western Blot Take out the gel
plates from cassette
Put the fiber pad on top black color side of casette and remove the
stacking gel
Put filter paper on top of fiber pad
Soak the fiber Wet resolving
pads and filter Put resolving gel on top of the filter paper in the cassette gel with buffer
paper in 1x
transfer buffer Put PVDF on top of resolving gel Transfer PVDF
membrane to
Put filter paper on top of PVDF 1x TB buffer

Put fiber pad on top of filter paper Soak PVDF

membrane in
Close and assemble the cassette into the cassette holder methanol

Place cassette holder into the electrophoresis tank

Stored the PVDF
membrane in 1x
Add 1x TB bufer fully into electrophoresis tank
TBST at 4oC
overnight Run electrophoresis at 100V for 90 minutes

Transfer the PVDF

membrane into 1x Stop the electrophoresis
TBST solution
Transfer the PVDF membrane into 5% skimmed
milk in 1x TBST

Shake the PVDF membrane into 5% skimmed

10 ml 5% milk in 1x TBST on shaker (1 hour, RT)
skimmed milk
Wash the PVDF membrane in 1x TBST by shaking (5 min)- (3x)
(in TBST)
Shake PVDF membrane in 5% skimmed milk (in TBST) and
1 ul anti β-
mouse anti-β-actin primary antibody mixture (1 hour)
actin antibody
Rinse the PVDF membrane with some 1x TBST
10 ml 5% Wash the PVDF membrane in 1x TBST by shaking (5 min)- (5x)
skimmed milk
(in TBST)
Shake PVDF membrane in 5% skimmed milk (in TBST) and goat
2 ul anti- anti-mouse IgG secondry antibody mixture (1 hour, RT)
mouse IgG
Rinse the PVDF membrane with some 1x TBST
antibody
Wash the PVDF membrane in 1x TBST by shaking (5 min)- (5x)

Keep the PVDF membrane with some 1x TBST

 Place the PVDF membrane onto the gel documentation system platform
Mix solution
A and solution Cover the PVDF membrane with the mixture
B at 1:1 ratio
(chemilumine Cover the PVDF membrane with transparent plastic sheet
scence ECL
reagents)
Detect signal with the gel documentation system

Diagram 3: Assembly of sandwich by fiber pad, filter paper, gel, PVDF, filter paper, and
fiber pad
RESULTS:
GROUP SDS PAGE WESTERN BLOT
1

3
 4

Table 1: Result of group 1,2,3,4 and 5 showing formation Comasie Blue stained targeted protein position on
the gel after SDS-PAGE and PDVF membrane in gel documentation system software for Western Blot.
 Experiment Manipulation Group
1 2 3 4 5
SDS Staining period 1 hour 1 hour 1 hour 30 mins 30 mins

Observation Clear and Clear and Clear and Unclear and faint Unclear
bright bright bright bands and faint
bands bands bands bands

Western Thickness of Thick Thin Thin Thick Thick

Blot Filter Paper

Washing TBST TBST Distilled TBST TBST

Solution Water

Primary 1:10000 1:5000 1:10000 1:10000 1:5000

antibody (2ul (4ul (2ul (2ul antibody) (4ul
Concentration antibody) antibody) antibody) antibody)
*(volume
antibody:
volume 5%
skimmed-milk)

Observation Visible Visible Visible Missing marker Missing

marker marker marker with unexpected marker
without without without band (lower with
bands bands bands molecular weight multiple
than expected) bands
Table 2: Result of SDS-PAGE and PDVF for group 1,2,3,4 and 5 with the manipulation on staining period,
thickness of filter paper, washing solution, primary antibody concentration and observation on the resulted bands

Diagram 4: Loading of Human Brain Endothelial cell sample and See-Blue Plus2 Pre-
Stained Protein On a NuPAGE 10% Bis-Tris Gel with MES SDS Running Buffer (Standard
Marker) of SDS for Satining with Coomassie Blue Stain
 Diagram 5: Loading of Human Brain Endothelial cell sample and See-Blue Plus2 Pre-
Stained Protein On a NuPAGE 10% Bis-Tris Gel with MES SDS Running Buffer
(Standard Marker) of SDS for Western Blot

For the experiment specifically on our Group (GROUP 4), the molecular weight of the single
band that form is below the expected molecular weight for targeted protein, Beta-actin.

198 kDa
98 kDa
62 kDa
Expected B-Actin
49 kDa Position:-
42 kDa Multiple Faint
38 kDa Protein Bands
28 kDa
Experimental Bright
Protein Position

Due to the missing marker ladder in Group 4 gel, comparison was made to the marker from
Group 2 as reference to for the protein molecular weight. The experimental protein position of
Group 4 is slightly lower than 28kDa but much higher than 17kDa. It is assumed around 25kDa.
 DISCUSSION:
1. Both immunoglobulin and antibody are components of the immune system.
Immunoglobulin and antibody comprised of glycoprotein molecules produced by plasma
cells that are disease-fighting proteins developed by most vertebrates in response to a
particular antigen. They act as a critical part of the immune response by specifically
recognizing and binding to particular antigens, such as bacteria or viruses, and aiding in
their destruction. Immunoglobulins are attached to the B cell membrane while antibodies
float in the circulation. The main difference between immunoglobulin and antibody is that
immunoglobulin has a transmembrane domain in order to be attached to the plasma
membrane whereas antibody does not have a transmembrane domain. There are five
antibodies produced by plasma cells and are classified by isotype, each of which differs in
function and antigen responses due to the amino acid sequence in the constant region (Fc)
of the antibody heavy chains structure. There are five antibody type in placental mammals:
IgA, IgD, IgE, IgG and IgM.

2. IgG is the major immunoglobulin in human blood serum, lymph fluid, cerebrospinal fluid
and peritoneal fluid and becoming a part of the secondary immune response to an
antigen/humoral immune response. IgG molecules are able to react with Fcγ receptors that
are present on the surface of macrophages, neutrophils and natural killer cells, and can
activate the complement system. IgG is produced in a delayed response to an infection and
can be retained in the body for a long time making it ideal for passive immunization by
transfer of this antibody. Because of its relative abundance and excellent specificity toward
antigens, IgG is the principle antibody used in immunological research and clinical
diagnostics.

3. There are four IgG subclasses described in human, mouse and rat. The subclasses differ in
the number of disulfide bonds and the length and flexibility of the hinge region.
Determination of IgG subclasses can be a valuable tool in indicating a potential antibody
deficiency and association with disease.
4. Actin play important roles in many cell functions such as maintenance of cytoskeleton, cell
motility, adhesion, endocytosis, exocytosis, secretion, signal transduction, and other
activities. Among its three main isoforms (α, β and γ), α-actin is normally found in smooth
muscle cells whereas β- and γ-actin isoforms are found in all cells. Higher vertebrates
(mammals and birds) express six different highly conserved actin isoforms that can be
classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-
CAA), 2) smooth muscle actins (SMAs),α-SMA and γ-SMA, and 3) cytoplasmic actins
(CYAs), β-CYA and γ-CYA.

5. One of six actin isoforms which is beta actin that is originated from cytoplasmic actin is a
highly conserved cytoskeletal protein involved in cell structure, motility, and cohesion.
They are also used as loading controls in protein assays. Beta-actin is known as a
“housekeeping” protein as it is expressed constantly, and present at high levels making it
is useful loading control in for Western blot analysis

6. Jurkat whole cell lysate sample is use as the source of beta actin protein which is detected
by beta actin primary antibody of IgG isotype. The purified beta actin protein shall be made
as positive control for separation by SDS-PAGE and subsequent Western blot analysis.

7. Sample preparation is a crucial step since in Western Blot, any antibodies will only
recognize proteins that have been reduced and denatured, because this reveals epitopes that
would otherwise be obscured by secondary and tertiary folding of the proteins. However,
some antibodies will only recognize epitopes on proteins in their native, folded state. The
sample is mixed with 5X loading buffer comprising the SDS,1M Tris buffer at pH 6.8.
bromophenol blue, B-mercaptoethanol, and glycerol. The Laemmeli reagent is use as
sample buffer for denaturing and loading of protein samples in SDS-PAGE.
8. To prepare the sample, each sample is treated with a number of reagents. Lysates are
prepared in denaturing buffer with reducing agents which is beta-mercaptoethanol (BME)
that function to dissolves disulfide bridges in the protein sample. The samples are also
boiled on the heat block to denature the proteins and dissociate any weak bonds. The
addition of these reagents ensures that the protein is broken up into smaller parts when it’s
run through the SDS-PAGE gel. A tracking dye, bromophenol blue, is additionally added
to the sample to allows visualisation of protein when it is pipetted into the well. For this
step, it is important that the protein is inserted into the right area, the well, instead of
between well or outside the well altogether. Next, the tracking dye enable monitoring the
electrophoresis when is complete. The tracking dye will travel down the gel faster than any
protein because it is smaller than any protein, and so when the dye hits the bottom it is an
indication to stop electrophoresis. Moreover, glycerol can be added to the sample to make
it denser so that the sample runs down the gel better.

9. The lysis buffer in which the loading buffer is diluted is RIPA reagent. RIPA buffer
function to lyses and extracts protein from cultured mammalian cells effectively by
extraction of membrane, nuclear and cytoplasmic proteins in all type of cell. Thus, this
eventually help in detection of beta actin targeted protein which is a type of cytoplasmic
actins (CYAs), β-CYA which also present in all type of cell including the Jurkat cell, a
type of lymphocyte cell.

10. Polyacrylamide gel electrophoresis (PAGE) is the method to separate and characterize our
targeted proteins which is Beta Actin from the Jurkat cell. Both SDS PAGE and Western
Blot are based on charge or pI (isoelectric point) separation. However, SDS PAGE not able
to confirm the presence of specific protein because proteins of different type with same
molecular weight can appear as bands. On contrary by detection in western blot, the
confirmation on the presence of blot can be done as the gel containing the protein is
transferred to nitrocellulose membrane. It is a type of confirmation test of expression level
of a protein using an antibody and has stronger validity than SDS page. In real life, Western
 blot is often used as a follow-up test to confirm the presence of an antibody and to help
diagnose a condition.

11. In this experiment, the method of choice which is SDS-PAGE electrophoresis is a type of
denaturing method as it treats the proteins with anionic SDS detergent
(sodiumdodecylsulfate). SDS, sodium dodecyl sulfate shall gives each protein negative
charge so that proteins only run based on size. Secondary- and tertiary structure may
destroyed during this process. Sodium dodecyl sulfate (SDS) is an amphipathic detergent
with a strong protein-denaturing effect and binds to the protein backbone at a constant
molar ratio. It has an anionic head and a lipophilic tail. It binds non-covalently to proteins
with the ratio of one SDS molecule per two amino acids. SDS causes denaturation and
disassociation of protein from each other without disturbing the covalent cross-linking.
SDS binds to the proteins and give negative charge. Thus, it masking the intrinsic charge
of a protein, leading to equally negatively charged proteins. This is done as SDS and the
reducing agent help to cleaves disulfide bonds to allow proteins unfold into linear chains
with negative charge proportional to the polypeptide chain length. Hence the results that
all proteins migrate toward the anode that is the positive charge electrode. SDS-treated
proteins have very similar charge-to-mass ratios, and similar shapes.

12. After SDS PAGE, a mixture of proteins is separated based on molecular weight, and thus
by type, through gel electrophoresis. These results are then transferred to a membrane
producing a band for each protein. Since, SDS-PAGE method separates protein on
molecular weight by using sodium dodecyl sulfate (SDS) which is an anionic detergent
applied to protein samples to linearize proteins and to impart a negative charge to linearized
proteins, the heating up of the samples during sample preparation may break up the
disulfide bonds of the protein giving out multiple bands. The multiple bands could be due
to the protein forming an oligomer that falls apart when the protein is denatured by SDS.
Besides, it maybe due to that the protein having multiple chains held together by disulfide
bonds that are reduced and broken by Mercapto-ethanol in the gel loading buffer.
13. Western Blot is an immunoassay test method that detects specific proteins in blood or
tissue by combining electrophoresis step and transferring (blots) the separated proteins onto
a membrane. Immunoblotting can be used to determine a number of important
characteristics of protein antigens such as the presence and quantity of an antigen, the
containing buffers. Additionally, unspecific binding is blocked before the addition of
specific antibodies. The immunodetection allow identification of specific antibodies after
the separation and blotting of proteins. Specific antibodies (mono- or polyclonal) bind to
proteins. Unspecifically binding antibodies are removed by washing with detergent.

14. The gel matrix made up of acrylamide gel polymer which is actually a polymer of
acrylamide and bis-acrylamide. Acrylamide forms linear polymers and bisacrylamide
make crosslinks between polyacrylamide chains. The pore size is determined by the ratio
of acrylamide to bisacrylamide, and by the concentration of acrylamide. The gel matrix can
easily be varied in density by adjusting the concentration of acrylamide. In fact, the top
part of the gel, known as the stacking gel, contains a lesser amount of acrylamide and
functions to concentrate the proteins above the lower gel, known as the resolving gel. The
resolving gel has greater amount of acrylamide to produce a higher resolution, or greater
distance, between each protein band in the sample.

15. A high ratio of bisacrylamide to acrylamide and a high acrylamide concentration cause low
electrophoretic mobility. The strength of the gel allows easy handling.

16. Polyacrylamide gel electrophoresis of SDS-treated proteins eliminates the influence of the
structure and charge, and proteins are separated solely based on polypeptide chain length.

17. The purpose of stacking gel is to make sure all the proteins start separating at approximately
the same time. The pore size is larger so that it is easier for large protein to move in order
 to catch up with the smaller protein. As the proteins are denatured and linearized by heating
and SDS the proteins are loaded onto the wells on the stacking gel. The denatured proteins
have a uniform mass to negative charge ratio. Since the current run from negative (top) to
positive (bottom), the proteins move toward the bottom. When the electricity is turned on,
the proteins and Tris-glycine enter the stacking gel. In stacking gel with pH 6.8, the N-
terminal amino group of the proteins and amino acids are protonated at equilibrium which
makes them less negative. The average electrophoretic mobility is very slow. A Glycine-
chloride ion boundary is formed since glycine moves slower than chloride ion. However,
glycine still runs slightly faster than other proteins. As a result, the glycine keeps pushing
the protein towards the chloride ion. In other words, the proteins are trapped between
glycine and chloride ion. The proteins form a very tight band inside the stacking gel.

18. As samples are being loaded in the wells at the top of the gel and current are started, not
all of the sample enters the gel at the same time. Thus the lower pH and low acrylamide
percentage in stacking gel will stack all of the proteins in sample into as narrow of a band
as possible, so that they all enter the resolving gel at essentially the same time. The stacking
gel concentrates proteins loaded into the sample wells so that they are resolved as a unified
line once they enter the stacking gel. If the stacking gel is missing, band of interest could
be very diffuse, and may not match up with molecular weight marker.

19. In SDS-PAGE, the stacking gel usually contains chloride ions which migrate faster than
proteins. While the buffer that the stacking gel was dissolved in, usually contains glycine
which migrates more slower than protein. Hence, this causes the protein to be sandwiched
in one band between the chloride and the glycine.

20. The stacking gel acts to create better resolution of the separating of the molecules. It does
this by creating an ionic gradient that traps the molecules. Negatively charged chloride
ions from Tris-HCl and positively charged glycine molecules create the gradient that keeps
 molecules moving together until they reach the separation gel in which pH changes that
allows the molecules to then be separated based on size.

21. The stacking gel also has a larger pore size than the resolving gel, making it loose enough
for large and small proteins can go through it at a similar rate. Electrophoresis is run at low
voltage during this period to ensure everything migrates toward the resolving gel slowly
and does not get separated. As the pores are smaller in the resolving gel, the size of the
protein determines the speed. So the smaller the protein is, the faster it will reach the
bottom.

22. Besides, Tris pH 6.8 is added to the stacking gel and Tris pH 8.8 is added to the resolving
gel to create a pH gradient. This pH gradient is also a vital component as it allows helps
the current run from negative cathode at the top of the resolving gel to the positive anode
at the bottom of the resolving gel. The reason for the lower pH is that, this "lower ionic
strength implies higher electrical resistance and consequently a higher electric field,
provoking the faster movement of the proteins and of every other charged particle in the
gel. Once the protein reaches the resolving gel, the pH changes from 6.8 to 8.8 and the
pores are smaller. As pH increases, the N-terminal amino groups are deprotonated, and the
sandwich is broken up. Amino acids and proteins are more negatively charged at
equilibrium than in stacking gel. As a result, glycine moves faster than proteins. Glycine
and chloride ions move ahead of the proteins. Then when the single band of protein hits
the resolving gel, voltage can be increased, and proteins will be separated by size as they
migrate through the smaller pores.

23. When the gel is in polymerization stage, a 10-well comb is placed in the top to produce
wells that can be used later for lanes to place sample into. These wells ensure that each
sample stays away from the others. One of the lanes is filled with marker followed by
sample loading into two wells alternately.
24. The gel is placed inside the electrophoresis chamber because the electrophoresis chamber
runs at 100V for 10 minutes through the gel, catalyzing the migration of the SDS-coated,
negatively charged proteins to the bottom of the gel over an hour period under 150 V.

25. Coomassie blue staining use for protein visualization. However, another better option can
be improvised is when choosing 2,2,2-trichloroethanol(TCE) to added to the mix for the
same purpose. This technique of TCE has distinct advantages over other methods of protein
visualization because it is quick, accurate to 0.2 micrograms of proteins, relatively cheap,
and allows for downstream applications with the gel. When TCE reacts with the
tryptophans in protein while under the presence of UV light it produces a fluorescent glow.
However, Coomassie blue staining is still a common method that is as accurate and
similarly priced as the TCE method but, it takes an hour to perform that is longer than the
TCE method. Other methods of protein visualization such as silver staining and
autoradiography are more accurate than TCE or Coomassie, but these methods are either
much more expensive or take much longer.

26. When analyzing an SDS-PAGE gel visualized using Coomasie Blue Staining, the markers
should be in the first or last lanes. Heavier proteins, will migrate a shorter distance and will
appear at the top of the gel while smaller proteins will appear lower on the gel. Actin, with
a molecular weight of 42kDa, should appear very close and in between to the 49-38 kDa
marker at the top of the gel. This proteins, in an ideal purification, should showcase a bright
actin band accompanied by dim bands of other proteins, which are just other remaining
Jurkat cell proteins. A brighter band indicates a greater relative purity while a dimmer band
indicates a lesser relative purity. The marker proteins provide an easy way to determine the
molecular weights of the proteins in each lane. Also, as Coomasie staining does not bind
specifically to actin but, other proteins of other molecular weight will appear also.
27. Primary antibodies are usually applied first, which are then recognized by a secondary
antibody. The secondary antibody is conjugated with colour, radioactivity or an enzyme
for detection. After immunodetection it is possible to strip the antibody off the membrane
for further analysis in order to detect other specific antibodies from the experimental
protein mixture.

28. During the SDS PAGE process, the manipulation on the period of staining with Coomassie
Blue Staining was done. Group 1,2 and 3 stain the gel for an hour while Group 4 and 5
stain for only 30 minute. Hence For the one hour stained gel produce clearer and high
intensity coloured bands compared with the 30 minutes stained gel that produce unclear
separation and faint bands.

29. Western Blot aim to transfer the Beta-actin proteins of Jurkat cell that was separated by
SDS-PAGE onto another membrane that can be used in the Western Blot for the following
lab. The SDS-PAGE gel is placed in a sandwich between blotting paper and a nitrocellulose
membrane. The nitrocellulose membrane has a very high affinity for proteins. Electrodes
are attached to the membrane-gel sandwich in a buffer solution with a negative charge at
the gel and a positive charge at the membrane. A perpendicular current is run through the
electrodes, catalyzing the migration of the negatively charged proteins to the positive
charge at the nitrocellulose membrane.

30. During the transfer of protein on the gel onto the transfer membrane, it is possible that
some of the SDS is washed out, and the protein partially re-naturates again by regains its
2D and 3D structure. However, the applied electric charge causes the proteins to travel out
of the gel vertically to the direction they traveled in on the gel, onto the membrane and
bind to it.
31. The transfer is done using an electric field perpendicular to the surface of the gel. Hence,
the proteins adhere to the membrane in the same pattern as they have been separated in
SDS PAGE due to interactions of charges. The sandwich arrangement comprised of fiber
pad placed at each end followed by filter papers to protect the gel and blotting membrane
at the centre. For precaution step, it is ensured that contact of gel and membrane is close
enough to ensure a clear image and ensure the membrane is place between the gel and the
positive electrode. The membrane must be placed as such, so that the negatively charged
proteins can migrate from the gel to the membrane. PVDF membranes is used because it
provide better mechanical support and allow the blot to be reprobed and stored. Washing
step is done thoroughly to prevent high background in the PVDF membranes.

32. Blocking is important to prevents antibodies from binding to the membrane

nonspecifically. Blocking is made with nonfat dried milk diluted in TBST to reduce the
background.

33. Skimmed milk is choose as blocking solution as it is inexpensive and easily available.
However, milk proteins are not compatible with all detection labels. Since milk contains
casein, which is itself a phosphoprotein and biotin. The phosphoprotein bind to anti-
phospho antibodies which causes non-specific binding and high background noise. Milk
may mask some antigens if they are in low abundance which would cause faint bands. If
faint bands occur then decrease concentration to 1% in blocking and antibody solutions or
substitute with BSA. Besides, Tris solution is function to can a stable pH from factors that
may shift the pH.

34. Washing is very important as it minimized background and removes unbound antibody but
not for a really long time to prevent signal is reduced. TBST Buffer is blocking buffers are
used to dilute the blocking agent and antibodies as well as washing. The same blocking
buffer for diluting the antibody that is used for the blocking step for optimization of assay.
TBS is used since alkaline phosphatase is used.They are composed of Tween 20 in the
 blocking buffer and antibody buffer is used to prevent weak non-specific binding. The
specific binding of antibodies is strong and should not be affected. However, this detergent
Tween 20 in TBS is prepared fresh prior to use since it promote microbial growth. The
same blocking buffer for diluting the antibody that is used for the blocking step for
optimization of assay.

35. The separated causative protein was added to the blot to be bounded by any antibodies.
Further detection using labeled antibodies will be interpreted by comparing with known
negative or positive control samples in the other lanes will determine the presence or
absence of the causative protein by comparing with a molecular weight standard of a
known composition.

36. In this experiment the primary antibody that is used is rabbit Beta-actin antibody which is
a polyclonal antibodies. A primary antibody is used for selective detection and specifically
bind the protein of interest. The host species of primary antibody extracted should be
different from the species of the sample. Hence, the primary antibody that is choose is from
rabbit species has reactivity toward human cell which is beta actin from Jurkat cell of
human brain extracted cell. The isotype used is IgG which has been subclassed into IgG1,
IgG2, IgG3 and IgG4. β-Actin Rabbit detects endogenous levels of total β-actin protein.
This antibody may cross-react with the γ-actin (cytoplasmic isoform). It does not cross-
react with α-skeletal, α-cardiac, α-vascular smooth, or γ-enteric smooth muscle isoforms.
This is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with
endogenous immunoglobulins in the sample. The immunogen used for this antibody shares
77% homology with Gamma actin/actin cytoplasmic. However, the cross-reactivity with
this protein has not been confirmed experimentally. By putting in BSA solution allows the
antibody to be reused if the blot does not give good result and experiment can be repeated.

37. A polyclonal antibody recognize multiple epitopes on the same antigen. Each of these
individual antibodies recognizes a unique epitope that is located on antigen. Each
 individual antibody in a polyclonal mixture is technically a monoclonal antibody.It is
advantageous to use polyclonal primary antibodies as such antibodies recognize several
epitopes can help amplify the signal from target protein with low expression level. This
makes these antibodies ideal for immunoprecipitation and chromatin immunoprecipitation.
Polyclonals are highly tolerant of minor changes as it is less sensitive to antigen changes
such as in occurance of slight denaturation, polymorphism, heterogeneity of glycosylation
than monoclonal antibodies. Multiple epitopes generally provide more robust detection.

38. Polyclonal antibodies are collected from multiple B cell clones that have been activated by
the immune response of an immunized animal such as goat, sheep, or rabbit. The animals
that immunized with the same antigen shall develop consistent immune responses which
result in similarity of polyclonal antibody production between batch preparations.

39. Multiple antibodies also will be generated against one protein antigen and a single
antibodies also could have been cross-react with another protein that contains the same
epitopes. This happen because serum may contains antibodies against so many antigens.
Hence, antibody specificity is typically very low with raw serum to detect our target protein
specifically without cross-reacting with non-specific proteins.

40. The secondary antibodies that is used and recommended as being tested previously by
research, is goat anti-rabbit IgG which is monoclonal. Secondary antibodies function to
bind with the primary antibody to assist in detection, sorting and purification of target
antigens. To enable detection, the secondary antibody must have specificity for the
antibody species and isotype of the primary antibody being used and generally is
conjugated. The type should be against the host species of the primary antibody that is
used. Since the primary is a rabbit monoclonal, it require an anti-rabbit secondary. This
monoclonal clonility helps prevent cross-reactivity from nonspecific proteins, resulting in
high specificity and low background signal. The isotype is IgG without specifically
subdivided as rabbit has only one IgG subclass.
41. Monoclonal secondry antibodies (mAbs) is used because to recognize a single epitope
within an antigen. Since mAbs are produced from a single B cell in the spleen or lymph
nodes of an immunized mouse, the resulting antibodies are all identical. In addition, they
recognize the same epitope of a specific antigen. Since B cells have a limited lifespan it
fused with a myeloma cell to create an immortalized B cell-myeloma hybridoma to provide
a constant supply of highly specific monoclonal antibody. Monoclonals are produced by
rat mouse monoclonals, rabbit and goat. However, mAbs are actually ideal for use as
primary antibody in an assay, or for detecting antigens in tissues. They produce low
background staining and lot-to-lot variation, and have low cross-reactivity. As such, they
provide reproducible results and ensure efficiency in affinity purification.

42. Since the blot is developed using ECL, HRP-conjugated secondary antibodies is used. The
secondary antibody is conjugated to an enzyme that is reactive toward the primary
antibody. The enzyme that catalyze chemiluminescent reactions is horseradish peroxidase
(HRP) that is detected by signal that is produced correspond to the location of the target
protein. Chemiluminescent detection occurs when energy from a chemical reaction is
released in the form of light. The chemiluminescent western blotting substrates are
luminol-based. For example, in the presence of HRP and peroxide buffer, luminol oxidizes
and forms an excited state product that emits light as it decays to the ground state. Light
emission occurs only during the enzyme-substrate reaction. So, once the substrate in
proximity to the enzyme is exhausted, signal output ceases.

43. Hence, for improvisation consideration when choosing primary and secondary antibodies
whereby a secondary antibodies not necessarily specific to the host species of tested cell.
However, it is depends on and different to the primary antibody. In this experiment since
human cultured cells is used, almost any secondary such as mouse, rabbit, goat, chicken,
and rat are plausible unless it was generated in human cells. Primary antibodies aspects
need to consider are the region of the targeted protein. Antibodies are generated by
 immunization of host animals with a variety of immunogenic substances including full-
length proteins, protein fragments, peptides, whole organisms (bacteria), or cells. Hence,
to detect a protein fragment or a specific isoform or region of the full-length protein, the
antibody use is raised against an immunogen that is identical to or contained within the
fragment or region. For cell surface protein on live cells, antibody that is used is against an
extracellular domain of the protein. Besides the clonility is one of the aspect to be
considered as polyclonal antibodies tend to be cheaper because they detect multiple
epitopes on your protein and are therefore less specific. Monoclonal are more expensive
and take longer to produce, but specific to a single epitope. However, monoclonal
antibodies are most useful if you are trying to identify specific isoforms or region of a full-
length protein as stated in.

44. For precaution step, the Mini-PROTEAN tetra system plates are initially cleaned with 70%
ethanol and Kim wipes in order to remove any existing proteins, and should never be
touched directly by hands to avoid contamination. Water is added to the gel during the
polymerization stage in the Mini-PROTEAN tetra system to prevent oxygen from entering
the gel. Prevent formation of air bubble during pipetting the gel in between the glass spread
as the oxidation will disturb the separation and movement of protein along the gel. The gel
should be pipetted gently in between the glass to prevent the air bubbles formation that also
causing the gel not polymerized well. Besides, the water that use for leaking test and that
also used to overlay the resolving gel need to be dry using fiter paper to prevent any water
bubble or artifact is introduce into the gel The gel should be pipetted gently in between the
glass to prevent the air bubbles formation that also causing the gel not polymerized well.

45. A disadvantage of western blotting (immunoblot) is that it is time-consuming (compared

to ELISA) and has a high demand in terms of experience of the experimenter. Additionally,
it requires optimizing the experimental conditions (i.e. protein isolation, buffers, type of
separation, gel concentration, etc).
46. To accurately determine protein expression and interpret Western blot results, it is
important to use loading controls. Beta-Actin (42 kDa) is commonly chosen as a loading
control due to its general expression across all eukaryotic cell types. Loading controls
purpose is to ensure the protein of interest has been correctly loaded equally across all
wells, that it is being transferred correctly, and that all reagents are functioning normally.
Actin is the most highly-conserved proteins with molecular weight of 42 kDa. Beta-actin
is known as a housekeeping protein and used as a loading control. It also use to normalize
the levels of protein detected by confirming that protein loading is the same across the gel.
Beta-actin is an isoform of actin proteins. There are six different but highly conserved actin
isoforms in vertebrates. Beta-actin is a major constituent of the contractile apparatus. The
pattern is consistent with each lot. Actin exists in two forms, filamentous (F-actin) and
monomeric (G-actin) in constant and dynamic equilibrium. Monomeric actin is soluble in
most buffer conditions while the filamentous is insoluble in triton/NP-40/tween and only
solubilizes in strong detergents such as SDS and partially at high pH. The the extraction
different amounts of actin twill reflect the total protein content by doing a total cell lysis in
SDS by boiling cells in Laemmli buffer.

47. From the Western Blot result, it is expected only one band should be visible since the
antibodies only bind to the protein of interest and the unbound antibody is washed off
leaving only the bound antibody to the protein of interest. The thickness of the band
corresponds to the amount of protein present. Thus doing a standard can indicate the
amount of protein present. However, the gel of Group 4 after western blot result a faint
band to appear without marker appear. Besides, the gel of group 5 produce multiple band
with also invisible marker. The gels of Group 1, 2 and 3 would give out no band but with
visible clear marker.

48. The manipulation been done on the thickness of filter paper is also manipulating with
group 1, 4, and 5 are using thick filter paper but group 2 and 3 are given thin filter paper
which then might effect the transferring step. An extra thick filter paper should have higher
capacity for blotting than thinner filter paper and prevent wave-like transfer of protein
 pattern onto PDVF. However from the experiment, the take up of protein into the PVDF
membrane from the gel is well transferred in all groups that use both thin and thick filter
paper without showing distinct differences.

49. Besides, manipulation been done on washing solution whereby group 2 are using distilled
water and the rest are using TBST solution. From the experiment, there is no relative
difference on the result of gel between group that using distilled water with TBST whereby
there is no high background result for group that using distilled water even there was no
detergent of Tween 20 present as how in TBST washing. Hence washing using different
solution did not give much effect on the gel and protein visibility.

50. Moreover, different concentration of primary antibody manipulation also was done
between group 2 and 5 with group group 1,3,4 whereby the 4ul and 2ul was used
respectively according to 5000 ul 5% skimmed milk and 1ul antibody in 5000ul 5%
skimmed milk preparation ratio. It is expected that higher primary antibody concentration
in group 1and 5 would give better protein resolution with more antibody–antigen binding
than lower primary antibody concentration used by group 2,3 and 4. In contrary group 2
result no bands instead of high primary antibody concentration and group 5 would give
brighter and most high resolution compared to other groups as being expected from a high
primary antibody concentration application. However the multiple band also being resulted
from group 5 showing unspecific binding that maybe also cause by too high concentration
of the primary antibody and need to be optimize in future. Apart from that, from group 4
that using low concentration primary antibody also would still result appearance of bands
although it is at lower molecular weight than expected beta-actin molecular weight. The
band in group 4 is however less bright than that is produce by group 5 due to primary
antibody concentration difference. For group 2 and 3 that showing no bands is maybe due
the low concentration manipulation as well as being influence by other factors.
51. Group 1,2 and 3 which shows no bands can also arise due to many reasons related to
antibody, antigen, or buffer used. If an improper antibody is used, either primary or
secondary, the band will not show. In addition, the concentration of the antibody should be
appropriate as well. The signal may not be visible in a very low concentration. Another
reason for no visible bands is the lowest concentration or absence of the antigen. In this
case, antigen from another source can be used to confirm whether the problem lies with the
sample or with other elements, such as the antibody. Moreover, prolonged washing can
also decrease the signal. Buffers can also contribute to the problem. It should be ensured
that buffers like the transfer buffer, TBST, running buffer and ECL are all new and
noncontaminated. If the buffers are contaminated with sodium azide, it can inactivate HRP.

52. For Group 4 gel that produce unusual or unexpected bands can be due to protease
degradation, which produces bands at unexpected positions. In this case it is advisable to
use a fresh sample which had been kept on ice or alter the antibody. The band is produce
at lower molecular weight than expected. There are several possibility that cause this
condition which are the target protein has been cleaved or digested, splice variants exist,
or there is another protein bearing the same/similar epitope has been detected by antibody.
This problem can be fix by using a fresh sample which has been kept on ice or by adding
fresh protease inhibitors to the lysis buffer. This blurry bands are also caused by high
voltage or air bubbles present during transfer. In this case, it should be ensured that the gel
is run at a lower voltage, and that the transfer sandwich is prepared properly. In addition,
changing the running buffer can also help the problem. The band also show faint coloured
or weak signals that is caused by low concentration of antibody or antigen. Increasing
exposure time can also help to make the band clearer. Another reason could be skimmed
milk masking the antigen. In this case use BSA or decrease the amount of milk used.

53. Group 5 gel result shows multiple bands at various molecular weights because primary
antibody concentration may be too high, or there is a cross-reactivity with similar epitopes
on other proteins. To handle this problem, an affinity-purified primary antibody can be
use. Besides, primary antibody concentration should be optimize, changing another
 antibody or check antibody specificity with blocking peptide. Furthermore, this multiple
band also maybe due to Secondary antibody concentration is too high which lead to non-
specific binding. Hence, the concentration of the secondary antibody should be optimize
or decrease. Using affinity-purified secondary antibody and repeat immunodetection with
secondary antibody alone to check for non-specific binding are also another suggested
alternative to prevent such problem from arising. This problem also may due to the protein
sample has multiple modified forms in vivo such as acetylation, methylation, myristylation,
phosphorylation, glycosylation. Hence, use an agent to remove modifications so that the
protein runs at the expected size. For bands are of lower molecular weight is maybe due to
the target in protein sample has been digested. Thus, sufficient protease inhibitors need to
incorporated into sample buffer. This problems may arising that due to the protein target
form multimers. Hence, to overcome it, try boiling in Laemmli buffer for 10 min rather
than 5 min to disrupt multimers.

54. During the detection of protein band using gel documentation system, the ECL that was
being use is expired and also the ECL was not applied to cover the PVDF membrane at the
right side. Hence, this causing no band and marker were being observed from the software.
Hence the improvisation should be done by making a small cut on the edge of resolving
gel prior to the transfer step to enable detection of back and front side of the gel and ECL
would be covered accordingly.

55. The data produced with a western blot is typically considered to be semi-quantitative
because it provides a relative comparison of protein levels, but not an absolute measure of
quantity.

56. Another recomeneded optimization step that can be done is by incubating primary antibody
overnight at 4°C in TBST in blocking agent instead of only one hour incubation. This can
produce high background or nonspecific bands appear. Besides, this also help to reduce
possibility of producing low signal whereby the protein of interest cannot be detected after
a short exposure of blot to film.
CONCLUSION:

The antibody could recognize our targettd β-actin protein from Jurkat cell of Human Brain
endothelial cell in western blot. The results in this SDS-PAGE of group 4 that was stained only
for 30 minutes give out faint coloured band compared to the other group that stain with Coomassie
Blue stain within an hour. However, the protein was separated well into several multiple bands.

For Western Blot, shows successful result as there is bands of Beta-actin produce from the
protein separation and transfer of protein from gel to PDVF membrane. However, the result from
blocking and application of primary and secondry antibody produce band that having different
molecular weight and not as expected to be which supposely 42kDa. The molecular weight of the
band is lower which is less than 28kDa marker band that is around 25kDa. The sample produce
less bright beta-actin band and a few faint multiple band. This result is maybe due effected by the
manipulation that was done which are using thick filter paper, washing using TBST buffer as well
as application of lower primary antibody concentration. Besides, the marker was also not appear
entirely as expected.
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