Download Gel electrophoresis

Gel electrophoresis
Gel electrophoresis is a method that
separates macromolecules-either nucleic
acids or proteins-on the basis of size,
electric charge, and other physical
properties.
Many important biological molecules such
as amino acids, peptides, proteins,
nucleotides, and nucleic acids, posses
ionisable groups and, therefore, at any
given pH, exist in solution as electically
charged species either as cations (+) or
anions (-). Depending on the nature of the
net charge, the charged particles will
migrate either to the cathode or to the
anode.
1
Electrophoresis
The term electrophoresis describes the
migration of charged particle under the
influence of an electric field.
Electro refers to the energy of electricity.
Phoresis, from the Greek verb phoros,
means "to carry across."
A GEL
A gel is a net. The suspended particles are
single large molecules or aggregates of
molecules or ions ranging in size from 1 to
1000 nanometers.
gel electrophoresis refers to the technique in
which molecules are forced across a span of
gel, motivated by an electrical current.
2
Activated electrodes at either end of the gel
provide the driving force.
A molecule's properties determine how
rapidly an electric field can move the
molecule through a gelatinous medium.
Agarose
There are two basic types of materials used
to make gels: agarose and polyacrylamide.
Agarose is a linear polysaccharide made up
of the basic repeat unit agarobiose. Agarose
is usually used at concentrations between
1% and 3%.
Agarose is very fragile and easily destroyed
by handling. Agarose gels have very large
"pore" size and are used primarily to
3
separate very large molecules with a
molecular mass greater than 200 kdal.
Agarose gels, the processed fast, But their
resolution is inferior. That is, the bands
formed in the agarose gels are fuzzy and
spread far apart. This is a result of pore size
and it cannot be controlled.
Preparation of Agarose
Agarose gels are formed by suspending dry
agarose in aqueous buffer, then boiling the
mixture until a clear solution forms. This is
poured and allowed to cool to room
temperature to form a rigid gel.
4
Polyacrylamide
polyacrylamide gel electrophoresis (PAGE)
involves separation of protein on the basis of
charge and molecular size.
The pore size of the gel may be varied to produce
different molecular sieving effects for separating
proteins of different sizes. In this way, the
percentage of polyacrylmide can be controlled in a
given gel. By controlling the percentage (from 3%
to 30%), precise pore sizes can be obtained, usually
from 5 to 2,000 Kdal. This is the ideal range for
gene sequencing, protein, polypeptide, and enzyme
analysis.
5
Gradient gels
It provides continuous decrease in pore size
from the top to the bottom of the gel, resulting
in thin bands. Polyacrylamide gels offer
greater flexibility and more sharply defined
banding than agarose gels.
Proteins
Proteins are important in the structure and
function of all living organisms. Some proteins
serve as structural components while others
function, defense, and cell regulation. Some
proteins serve as enzymes that act as biological
catalysts which control the biochemical events.
Amino Acids
The fundamental unit of protiens is the amino acid.
Each amino acid contains an amino group (-NH2)
and a carboxylic group (-COOH) attached to a
central carbon called the alpha carbon. A R-group
6
(or side chain) is also attached to the alpha carbon.
The R-group, or side chain, determines the nature
of different amino acids.
Twenty amino acids have been identified as
constituents of most proteins. These amino acids
differ from each other in the nature of the R-group
attached to the alpha carbon. The identity of the
particular amino acid depends on the nature of the
R group.
Classification of Amino Acids
Amino acids are classified according to properties
of the R groups. The first of these depends on the
polar or nonpolar nature of the side chain. The
second depends on the presence of an acidic or
basic group in the side chain. Another criteria that
7
is taken into account is the presence of functional
groups in the side chains and the nature of those
groups.
Electrophoresis of Proteins
Proteins can be separated and purified by
electrophoresis. Methods for separating
proteins take advantage of properties such as
charge, size, and solubility, which vary from
one protein to the next. Because many proteins
bind to other biomolecules, proteins can also
be separated on the basis of their binding
properties. The source of a protein is generally
tissue or microbial cells. The cell must be
broken open and the protein must be released
into a solution called a crude extract. If
necessary, differential centrifugation can be
used to prepare subcellular fractions or to
isolate organelles. Once the extract or
8
organelle preparation is ready, a variety are
available for separation of proteins. Ionexchange chromatography can be used to
separate proteins with different charges
(similar to the way amino acids are separated).
Other chromatographic methods take
advantage of differences in size, binding
affinity, and solubility. Nonchromatographic
methods include the selective precipitation of
proteins with salt, acid, or high temperatures.
In addition to chromatography, another
important set of methods is available for the
separation of proteins, based on the migration
of charged proteins in an electric field, a
process called (gel) electrophoresis. Gel
electrophoresis is especially useful as an
analytical method. Its advantage is that
proteins can be visualized as well as separated,
permitting a researcher to estimate quickly the
number of proteins in a mixture or the degree of
9
purity of a particular protein preparation. Also, gel
electrophoresis allows determination of crucial
properties of a protein such as its isoelectric point
and approximate molecular weight.
Amino acids differ not only in R-group
characteristics but also in molecular weight.
Different amino acids are linked together in a
linear chain by peptide bonds in various
combinations and sequences to form specific
proteins. The net charge of a protein will
depend on its amino acid composition. If it has
more positively charged amino acids such that
the sum of the positive charges exceeds the
sum of the negative charges, the protein will
have an overall positive charge and migrate to
the cathode (negatively charged electrode) in
an electrical field. Proteins even with a
variation of one amino acids will have a
different overall charge, and thus are
electrophoretically distinguishable.
10
polypeptides that make up complex proteins.
The break up of complex proteins into their
respective polypeptides allows us to study the
structure of proteins that result from the
interaction of several genes.
A gene is a discrete unit of hereditary
information that usually specifies a protein. A
single gene provides the genetic code for only
one polypeptide. Thus, a protein consisting of
four polypeptides requires the interaction of
four genes to synthesize that specific protein.
A molecular weight protein marker is used to
prepare a standard separation curve with which
various unknown proteins or polypeptide
fractions can be identified.
11
Nucleic Acids
The double helix structure originally
proposed by Watson and Crick is the most striking
feature of DNA structure. The two coiled strands
run in antiparallel directions and are held together
by hydrogen binds between complentary bases.
Adenine pairs with thymine and guanine with
cytosine. Eukaryotic DNA is complexed with
histones and other basic proteins, while prokaryotic
DNA occurs in "naked" form not complexed to
proteins.
Nucleic acids transmit hereditary information and
determine which proteins a cell manufactures.
There are two classes of nucleic acids found in
cells: ribonucleic acids (RNA) and
deoxyribonucleic acids (DNA). DNA comprises
12
the genes, the hereditary material of the cell and
contains instructions for making all the proteins
needed by the organism. RNA functions in the
process of protein synthesis. Nucleic acids are
large, complex molecules. Their name --nucleic
acid-- reflects that they are acidic and were first
identified in nuclei.
Nucleic acids are made of only four nucleotides in
a regular and an interesting arrangement. Beadle et
al experiments had shown that genes control the
production of enzymes, which are proteins.
Nucleic acids are polymers of nucleotides,
molecular units that consist of the following:
1. a five-carbon sugar, either ribose or
deoxyribose,
2. a phosphate group, and
13
3. a nitrogenous base, a ring compound
containing nitrogen.
The nitrogenous base may be either a double-ringed
purine or a single-ringed pyrimidine. DNA
commonly consists of


Purines
o adenine (A)
o guanine (G)
Pyrimidines
o cytosine (C)
o thymine (T)
with the sugar deoxyribose and phosphate. RNA
commonly consists of


Purines
o adenine (A)
o guanine (G)
Pyrimidines
o cytosine (C)
o uracil (U)
with the sugar ribose and phosphate.
The removal of the phosphate group from a
nucleotide yields a compound termed a nucleoside,
composed of a base and a sugar.
14
Nucleic acid molecules are made of linear chains of
nucleotides. The nucleotides are linked together by
covalent bonds to each other.
The specific information of the nucleic acid is
coded in the unique sequence of the four kinds of
nucleotides present in the chain. DNA is composed
of two nucleotide chains entwined around each
other in a double helix.
The base sequence of nucleic acids can be
determined in a manner similar to determination of
the amino acid sequence of protein, but it is more
efficient, particularly for DNA, to use specialized
techniques. Restriction endonucleases can be used
to cleave DNA molecules into fragments of
suitable size. In a direct chemical method, four
samples of a given restriction fragment can each be
treated with a selective reagent, causing cleavage at
a given base. The resulting mixtures can be
analyzed by gel electrophoresis, which separates,
15
on the basis of size, the oligonucleotides produced
by this treatment. The base sequence of the
oligonucleotide can be "read" directly from the
sequencing gel.
16