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Lab Report Sds-Page WB - PT 1 (1-5)
Lab Report Sds-Page WB - PT 1 (1-5)
1 Introduction
The technique of electrophoresis which has been studied for over two centuries involve the
movements and separation of charged particles such as DNA and proteins, in a liquid medium
under the action of an electric field for the purpose of investigating the subunits composition,
verifying homogeneity of samples and purification process to name a few. Sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE), is the most widely used method to
separate proteins based on molecular weight, enabling determination of sample protein
composition, purification of proteins, presence of target of interest in the sample and even
structural characteristics (Rockland, 2018). The critical steps primarily employed in SDS
PAGE comprises of gel preparation, sample preparation, electrophoresis, protein staining
(or western blotting) and analysis of the generated banding pattern. In this experiment, SDS-
polyacrylamide gel electrophoresis (SDS-PAGE methods were used to resolve different
proteins in a HCEC lysate and in combination with western blot, to determine the presence of
a Beta-actin protein in a Human Cerebral Endothelial Cell (HCEC) lysate. The HCEC lysate
are treated with SDS detergent and using PAGE the proteins are separated. Beta-actin is then
identified through immunoblotting techniques. Actins, one of the most highly-conserved
proteins are comprised of three main isoform groups, alpha, beta and gamma. The alpha actins
are found in muscle tissues and are a major component of the contractile apparatus while 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 (42 kDa) is also known as a
“housekeeping” protein as it is expressed constantly at high levels in all cell types used in
protein research. Due to that attribute, it is commonly chosen as a loading control in Western
blot analysis (Bio-Rad, 2018). During electrophoresis through a gel matrix, smaller proteins
will progress faster due to less resistance from the gel matrix. Other factors that influences on
the rate of migration of proteins through a gel matrix besides size include the structure and
charge of the proteins. SDS-PAGE technique eliminates the influence of the structure and
charge enabling protein separation solely based on polypeptide chain length.
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SDS is a strong anionic detergent that is present in the SDS-PAGE sample buffer. Initially,
samples are treated by boiling for 5 minutes with SDS and a reducing agent (normally
DTT(dithiothreitol) or B-ME(Beta-mercaptoethanol) to break down disulphide bonds holding
the tertiary and quaternary structure of proteins. SDS in turn binds strongly and denatures
proteins. This brings the folded proteins down to a rod shaped structure with a series of
negatively charged SDS molecules along the polypeptide chain. The negative charge conferred
to the amino acids gives a constant charge to mass ratio to all proteins allowing separation to
occur solely based on size (Rockland, 2018).
Figure 1: SDS and a reducing agent that cleaves disulphide bonds critical for proper folding,
forces proteins to unfold into linear chains with negative charge proportional to the polypeptide
chain length.
Additionally, the sample buffer contains tracking dyes such as Bromphenol blue for monitoring
electrophoresis run and sucrose/glycerol to increase sample solution density for ease of loading.
Once sample is loaded for PAGE, current is applied to the gel until tracking dye reaches the
bottom of the gel. After separation by electrophoresis, proteins can be bound to membranes
where they are fixed and readily accessible for immunological or biochemical analyses,
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however may lead to the formation of undesirable higher background and cross-reactivity.
Monoclonal antibodies in turn consist of homogeneous cloned immunoglobulin molecules
that bind to a single epitope within a target antigen. These antibodies are made by identical
immune cells that are all clones of a unique parent cell and produced by fusing antibody
producing cells from the spleen of the immunized animal (usually a rat or mouse) with an
immortalized cell line to produce single specificity antibodies. Immunodetection steps
mainly involve application of primary antibodies first, which are then recognized by a
secondary antibody. The secondary antibody in turn is conjugated with colour, radioactivity or
an enzyme for detection. The most commonly used enzymes are Horse Radish Peroxidase
(HRP) and Alkaline Phosphatase (AP). After immunodetection it is possible to strip the
antibody off the membrane for further analysis with other antibodies. Analysis of the western
blot can be carried out using a variety of different imaging systems (e.g. luminescence, color
reaction, autoradiography) (Johnson, 2018). Chemiluminescence, for instance is a popular
detection method that involves a chemical reaction occurring between an enzyme e.g.
horseradish peroxidase (HRP) and a chemiluminescent molecule such as luminol resulting in
a light emission. This light emission can be detected with a CCD imager. Chemifluorescence
attaches a fluorescent molecule to either the secondary or tertiary antibody which requires
excitation via a light source. Fluorescence involves using secondary antibodies conjugated with
a fluorescent molecule (fluorophore). Fluorophore excited by a light source releases of photons
which is then detected in the form of light. The light emitted from fluorophores is consistent
and directly proportional to the amount of protein on the membrane. Image analysis can then
be performed as the last step, using CCD camera based imaging or X-Ray film (Syngene,
2018).
2 Objectives (Aims)
2.1 Protocol I – SDS-PAGE
1. To prepare gel for SDS-PAGE.
2. To resolve different proteins in a Human cerebral endothelial cell (HCEC) lysate using
SDS-PAGE.
2.2 Protocol II –WESTERN BLOTTING
1. To perform protein transfer from gel to a solid-phase membrane support.
2. To identify β-actin protein (control protein) in the HCEC lysate by immunodetection.
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Glass plate and a spacer plates were wiped clean with 70% alcohol.
A small amount of vaseline were rubbed to the side of the glass plate
and both plates were brought together.
The glass plates were placed into the plate holder and clamped
Distilled water was pipetted in between the plates until full to check
for any leakage before being removed. The plated were dried using
filter paper.