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Abstract
SDS – Page or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is a method that
is used to study proteins. It is a technique and process widely used in forensics, genetics,
biotechnology and molecular biology to separate the protein molecules based on their
electrophoretic mobility. A SDS page proteins are separated in a poly acrylamide gel that
based on the molecular weight. This method were initially denature the proteins that
undergo electrophoresis, covalent structural features and properties of proteins can be
determined by SDS - Page , all the functional properties were destroyed includes the non
covalently metal ions. Example preparation is loading a buffer that contains SDS beta
mercaptoethanol , bromophenol blue and glycerol is being added to the sample protein that
may be derived from the virus tissues or prokaryotic tissues and purified proteins. Then once
sample is being heated at 95 degree Celsius in 5 minutes a large biomolecules proteins
consisting of one or more long chain of amino acids that contain a carboxyl group and amine
functional groups is ready to denature . SDS is anionic surfactant that contain polar head
group that have a negative charge at the end of a long chain hydrophobic carbon chain . It
will also denature native proteins by disturbing and suppressing the hydrogen bonds
hydrophobic and ionic interactions, while the reducing agent beta mercaptoethanol is used
to cleave the disulfide bonds and protein mixture is heated so SDS binds uniformly to the
protein. In SDs
Introduction
Electrophoresis is the electrophoretic separation of proteins. Complex protein mixtures
(from cells, column fractions, subcellular fractions, or immunoprecipitates) can be separated
by electrophoresis, which is also used to examine the component compositions of proteins
and confirm the homogeneity of protein samples. Additionally, it can be used to purify
proteins for later usage. Proteins move through perforations in a polyacrylamide gel matrix
during polyacrylamide gel electrophoresis in response to an electrical field; pore size shrinks
as acrylamide concentration rises. The protein migrates at a rate that depends on the pore
size, protein shape, size and charge.
According to the SDS-PAGE principle, when a charged molecule is being exposed to an
electric field, it will migrates towards the electrode carrying the opposite charge. The
relative mobility of charged species affects how the charged molecules are separated.
Due to decreased resistance during electrophoresis, the smaller molecules migrate
more quickly.
SDS, also known as sodium dodecyl sulphate, is a powerful detergent in high
proportions in the buffer used to prepare samples for electrophoresis. Cell membranes
must be lysed, and all proteins must be solubilized by SDS before samples like cells
may be run on a protein gel.
SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some
reducing agents function to break the disulphide bonds of proteins disrupting the
tertiary structure of proteins.
In Electrophoresis.
The gel cassette is removed from the casting stand and placed in the electrode assembly. The
electrode assembly is fixed in the clamp stand.1x electrophoresis buffer is poured in the
opening of the casting frame to fill the wells of the gel. Pipette 30ml of the denatured sample
in the well. The tank is then covered with a lid and the unit is connected to a power supply.
The sample is allowed to run at 30mA for about 1 hour. The bands are then seen under UV
light.
In Gel Staining.
In gel staining , Coomassie blue is used in this steps to stain proteins . The dye helps the
visualization of proteins in the form of blue bands after using methanol and acidified acetic acid
proteins . And the markers in a separate lane is used to determine the molecular mass of specific
molecules by comparing the distance they traveled relative to the marker.
RESULTS
Proteins of resultant isolates, i.e., sample one protein isolates (SPI) is characterized
for its molecular weight using sodium dodecyl sulfate polyacrylamide gel
electrophoresis. The electropherogram for Sample one and the reference standard
are illustrated in Figure below. Respective isolates were recorded ranging from 18 to
120 kDa. The electropherogram also presented numerous fractions having low
molecular weights. Moreover, SPI included several polypeptide bands ranging from
15 to 120 kDa, while FPI bands ranged between 42 and 86 kDa. Furthermore, the CPI
bands ranged from 7 to 120 kDa with fewer bands than other tested protein isolates.q
A trivial difference in the movement was observed in electrophoretic bands of SPI, FPI, and
CPI. Certain variations may be attributed to structural as well as compositional changes in
proteins along with their interaction with salts.
Once the proteins are in the running gel, they begin to separate because larger proteins tak
e longer to pass through the porous acrylamide gel than smaller proteins do. By adjusting t
he acrylamide concentration, you can change the size of the holes in the gel to match the siz
e of the proteins you want to separate.
Discussions
The effect of SDS page is to break down protein by disrupting disulfide bonds and tertiary
structures of the proteins. In which bring a folded parts of the protein to give off linear
molecules. We found that in putting a SDS page in some samples taken from the prokaryotic
or other viral structures will determine the protein information by broken it’s linkage. And
also when the gel remove from the glass plates it is ready for the thorough investigation and
research. It will now determined a sample molecular weight , used to determined specific
sizes of the protein , peptide mapping and it used to determined the purity of the proteins,
and analyze the different subunits of polypeptide subunits.
Conclusions
According to the results which mentioned for the first time using SDS-
PAGE for some specific samples , we can conclude that, researchers can use
protein analytes to analyze the structures and purity of the proteins. Even it
involves meticulous process , the best results always give off positive
outcome in which all the specific sample yields standardized identification
of molecular weight, and different unique polypeptide composition with
different structures. SDS Page is an method that is relevantly used by many
biochemists and chemists to study structures of proteins.
Acknowledgement
I would like to acknowledge my parents who help me to support my financial needs in
creating this paper . And also to my instructor Ms. Caburatan who help me in dong this
paper by giving some important format and synthesizing information in making this paper
. And also I would like to extend my gratitude to almighty one who give me strength and
wisdom to make this paper successful .
Literature cited