M- PHARMA [QA]
A technique for separating the compound of a
mixture of charged molecular (proteins , DNA
,RNA) in an electric field within a gel or other
The movement of electrically charged
molecular is an electric field often resulting
in their separation
It is a technique used for the separation of
Deoxyribonucleic acid, Ribonucleic acid or protein
molecules according to their size and electrical
charge using an electric current applied to a gel
What is a gel?
Gel is a cross linked polymer whose
composition and porosity is chosen based on
the specific weight and porosity of the target
Charged molecules are separated based on their
electrical charge and size.
T YPE OF GEL
A highly purified uncharged polysaccharide
derived from agar.
Used to separate macromolecules such as
nucleic acids, large proteins and protein
It is prepared by dissolving 0.5% agarose in
boiling water and allowing it to cool to 40 C.
Commonly used components: Acrylamide
monomers, Ammonium persulphate,
These free radicals activate acrylamide
monomers inducing them to react with other
acrylamide monomers forming long chains.
Used to separate most proteins and small
oligonucleotides because of the presence of
MATERIAL REQUIRED FOR GEL
Gel casting tray
Staining agent (dye)
Sample to be separate
GEL COSTING TRAYS
available in a variety of
sizes and composed of
The open ends of the
trays are closed with
tape while the gel is
being cast, then
removed prior to
voltage, rate of migration
The higher the voltage, the more quickly the
But if voltage is too high, gel melts
The best separation will apply voltage at no
more than 5V/cm of gel length.
During electrophoresis water undergoes
hydrolysis : H 2 O H + OHBuffers prevent the pH from changing by
reacting with the H+ or OH- products
Most common buffer used is called TRIS
Another compound is added to make Tris an
effective buffer — either boric or acetic acid
Another compound is added to bind metals
The buffer is either TBE or TAE
TBE is made with Tris/Boric Acid/EDTA
TAE is made with Tris/Acetic Acid/ EDTA
The standard concentration
used in staining DNA in
gels is 0.5-1ug/mL
Ethidium bromide is a
fluorescent dye that
intercalates between bases
of nucleic acids and allows
very convenient detection
of DNA fragments in gels.
Inserting itself between the
base pairs in the double
STAINING OF DNA
UV absorbance maxima at 300 and 360 nm and
emission maxima at 590 nm.
Detection limit of bound DNA is 0.5 -5 ng/band.
ethidium bromide is mutagenic so care must be
taken while handling the dye.
Other alternatives for ethidium bromide :
A comb is placed in the
liquid agarose after it
has been poured
Removing the comb
from the hardened gel
produces a series of
wells used to load the
METHOD FOR ELECTROPHORESIS
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix
Pour into casting tray with comb and allow to
Add running buffer, load samples and marker
Run gel at constant voltage until band separation
View DNA on UV light box and show results
Electrophoresis is employed in biochemical
and clinical field.
In the study of protein mixtures
Antigen antibody reactions
In fractioning protein.
In analysis of lipoprotein
Analysis of PCR products, e.g. in molecular
genetic diagnosis or genetic fingerprinting
Separation of organic acid, alkaloids,
carbohydrates, amino acids, alcohols,
phenols, nucleic acids, insulin.
In food industry
In combination with autoradiography