Agarose Gel Electrophoresis

o Agarose gels are commonly used to sort DNA and RNA molecules based on size. The
agarose gel concentration can be varied, based on the size of the molecules that need to be
isolated.
SDS-PAGE Electrophoresis
o Sodium dodecyl sulfate - polyacrylamide gel electrophoresis is used to separate proteins
based on size. The proteins are unfolded, or denatured, using SDS detergent, and run on a
polyacrylamide gel.
DNA Sequencing Gels
o Denatured DNA can be run on polyacrylamide gels, which allows scientists to determine the
sequence of the molecule.
Native Protein Electrophoresis
o Proteins can remain folded in the native conformation and run on gels to separate them by
both mass and charge.
Electrofocusing Electrophoresis
o Electrofocusing separates proteins on the basis of charge as well as pH; the gel used in this
type of electrophoresis has a pH gradient.












Theory


PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate
components of a protein mixture based on their size. The technique is based upon the principle
that a charged molecule will migrate in an electric field towards an electrode with opposite
sign.The general electrophoresis techniques cannot be used to determine the molecular weight of
biological molecules because the mobility of a substance in the gel depends on both charge and
size. To overcome this, the biological samples needs to be treated so that they acquire uniform
charge, then the electrophoretic mobility depends primarily on size. For this different protein
molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so
that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered
by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will
migrate towards the anode (positively charged electrode) are separated by a molecular sieving
effect based on size. After the visualization by a staining (protein-specific) technique, the size of
a protein can be calculated by comparing its migration distance with that of a known molecular
weight ladder(marker).

Gel electrophoresis is a method for separation and analysis of macromolecules
(DNA, RNA and proteins) and their fragments, based on their size and charge.
It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially
size independent) and in biochemistry and molecular biology to separate a mixed population
of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge.
[1]
Nucleic acid molecules are separated by applying an electric
field to move the negatively charged molecules through an agarose matrix. Shorter molecules
move faster and migrate farther than longer ones because shorter molecules migrate more easily
through the pores of the gel. This phenomenon is called sieving.
[2]
Proteins are separated by
charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis
can also be used for separation of nanoparticles.
Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during
electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the
thermal convection caused by application of the electric field, and can also act as a sieving
medium, retarding the passage of molecules; gels can also simply serve to maintain the finished
separation, so that a post electrophoresis stain can be applied.
[3]
DNA Gel electrophoresis is
usually performed for analytical purposes, often after amplification of DNA via PCR, but may be
used as a preparative technique prior to use of other methods such as mass
spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further
characterization.