Gel Electrophoresis

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins
according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by
an electrical field through a gel that contains small pores. The molecules travel through the pores
in the gel at a speed that is inversely related to their lengths. This means that a small DNA
molecule will travel a greater distance through the gel than will a larger DNA molecule.
As previously mentioned, gel electrophoresis involves an electrical field; in particular, this field
is applied such that one end of the gel has a positive charge and the other end has a negative
charge. Because DNA and RNA are negatively charged molecules, they will be pulled toward the
positively charged end of the gel. Proteins, however, are not negatively charged; thus, when
researchers want to separate proteins using gel electrophoresis, they must first mix the proteins
with a detergent called sodium dodecyl sulfate. This treatment makes the proteins unfold into a
linear shape and coats them with a negative charge, which allows them to migrate toward the
positive end of the gel and be separated. Finally, after the DNA, RNA, or protein molecules have
been separated using gel electrophoresis, bands representing molecules of different sizes can be
detected.
(source: http://www.nature.com/scitable/definition/gel-electrophoresis-286)

Gel electrophoresis

Gel electrophoresis is used to separate macromolecules like DNA, RNA and proteins. DNA
fragments are separated according to their size. Proteins can be separated according to their size
and their charge (different proteins have different charges).

How are DNA fragments separated using gel electrophoresis?

A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively
charged, it can be pulled through the gel by an electric field. Small DNA molecules move more
quickly through the gel than larger DNA molecules.
The result is a series of bands, with each band containing DNA molecules of a particular size.
The bands furthest from the start of the gel contain the smallest fragments of DNA. The bands
closest to the start of the gel contain the largest DNA fragments.

When is gel electrophoresis used to separate DNA fragments?

Gel electrophoresis can be used for a range of purposes, for example:

To get a DNA fingerprint for forensic purposes

To get a DNA fingerprint for paternity testing

To get a DNA fingerprint so that you can look for evolutionary relationships
among organisms

To
check
a
PCR
reaction.
Get video clip: Using gel electrophoresis to check a PCR reaction

To
test
for
genes
associated
with
a
particular
disease.
Get information sheet: Gel electrophoresis can be used to find genes
associated with a disease

When is gel electrophoresis used to separate proteins?

Thanks to TV shows like CSI, many people are familiar with the use of gel electrophoresis to
separate macromolecules like DNA. However, gel electrophoresis can also be used to separate
out proteins.
Different proteins have different sizes, mainly due to the number of amino acid building blocks
in their structure. Chemical modifications attached to the protein also affect its size. Different
proteins also have different charges. This can result from both the types of amino acid used to
construct them, as well as the types of modifications attached to them.
Different types of electrophoresis gels are used to provide different types of information. The
type of gel you choose therefore depends on the type of question you are asking.
Size Separation

Protein Electrophoresis
Typically, gels made from polyacrylamide are used to separate proteins on the basis their
different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in order
to unravel the protein. SDS is a detergent that gives all the proteins the same overall negative
charge so that when an electric current is applied to the gel, separation is only due to the size of
the protein. This technique is called SDS-PAGE (SDS-Polyacrylamide gel electrophoresis).

Small protein molecules move more quickly through the gel than larger proteins, resulting in a
series of bands. Each band contains a protein of a particular size. These can be compared with
standards of known sizes.
An SDS-PAGE gel has been used to separate proteins on the basis of size. The samples are the
blood of various shark species. The first lane contains markers of known sizes. Large proteins are
at the top of the gel and small proteins are at the bottom.
This technique might be used for many purposes, including purifying a particular protein, for
example to isolate an enzyme for the food industry.
Charge and pH separation

Isoelectric focussing (IEF) and agarose gel electrophoresis are two ways that proteins can be
separated by their different electrical charges. Unlike SDS-PAGE, the proteins are usually kept in
their native (folded) state. The type of gel that is used, and the solution around the gel, are also
different.
In agarose gel electrophoresis, proteins are loaded in the middle of the well. Those with a strong
negative charge move fastest towards the positive side of the gel, whereas positively charged
proteins move in the opposite direction.
This technique might be used to separate proteins that have the same molecular weight but
different charges, or when size is not important (e.g. to look at changes in the presence of
different protein during the development of a disease).
Two-dimensional electrophoresis

These days, charge (IEF) and size (SDS-PAGE) separation are often employed together in twodimensional electrophoresis, where charge separation is first used, and then these separated
proteins are separated on the basis on size.
This is a very effective method for identifying a particular protein from a tissue that may contain
thousands of proteins and where there may only be small differences between control and treated
samples (e.g. to look for a protein involved in resistance to insect predation in plants).
(source: http://www.biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis)

Electrophoresis is a technique used to separate and sometimes purify macromolecules especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it
is one of the most widely-used techniques in biochemistry and molecular biology.

When charged molecules are placed in an electric field, they migrate toward either the
positive or negative pole according to their charge. In contrast to proteins, which can have
either a net positive or net negative charge, nucleic acids have a consistent negative charge
imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or "gel".
Most commonly, the gel is cast in the shape of a thin slab, with wells for
loading the sample. The gel is immersed within an electrophoresis buffer that
provides ions to carry a current and some type of buffer to maintain the pH at
a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of
which have attributes suitable to particular tasks:
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5
to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy
to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the
gel. It is also non-toxic.
Agarose gels have a large range of separation, but relatively low resolving power. By
varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be
separated using standard electrophoretic techniques.
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is
dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.
Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because
oxygen inhibits the polymerization process, they must be poured between glass plates (or
cylinders).
Acrylamide is a potent neurotoxin and should be handled with care! Wear
disposable gloves when handling solutions of acrylamide, and a mask when weighing out
powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be
handled with gloves due to the possible presence of free acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power.
In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp.
However, under appropriate conditions, fragments of DNA differing is length by a single base
pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for
separating and characterizing mixtures of proteins.
(Source:
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/principles.html)