electrophoresis

By- devanshi jadaun

INTRODUCTION
Electrophoresis is the study of movement of charged particles in an
electric field.
Biomolecules like DNA, RNA, amino acids and proteins etc. carry positive
and negative charge with them.
TYPES OF ELECTROPHORESIS
Zone Electrophoresis
a) Paper Electrophoresis
b) Gel Electrophoresis
c) Cellulose acetate Electrophoresis
Free Electrophoresis
a) Micro Electrophoresis
b) Moving boundary
c)Capillary Electrophoresis
TECHNIQUES OF ELECTROPHORESIS
1. PAPER ELECTROPHORESIS :-Paper Electrophoresis is one of the type
of zone electrophoresis.
Principle:- 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 towards the anode.
EQUIPMENTS:-electrophoresis consist a basically of two items
1.Power Pack
2.Electrophoretic cell
TYPES OF PAPER ELECTROPHORESIS:-
1.Horizontal Paper Electrophoresis
2.Vertical Paper Electrophoresis
WORKING:-
1) A long strip of filter paper is moistened with a suitable buffer solution of the
desired p H and the sample is applied transversely across the central part of the
strip.
2) Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes.
3)Electric field of about 20 volts/cm is established.
4)The charged particles of sample migrate along the strip towards respective
electrodes of opposite polarity, according to net charges, sizes and interactions
with the solid matrix.
5)Homogeneous group of particles migrate as a separate band
6)The electrophoresis is carried out for 16-18 hours. 7)Proteins are stained
(bromophenol blue) to make them visible
8) The separated proteins appear as distinct bands.
2. GEL ELECTROPHORESIS:-
Principle :-
Gel electrophoresis involves an electrical field.
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
Types of gel electrophoresis:-
1.AGAROSE GEL:-
Poured Horizontally
Separate large molecules
Non-toxic
Mostly used for DNA separation
2.POLYACRYLAMIDE GEL:-
Poured Vertically
Separate small molecules
neurotoxin
Used for DNA or Protein separation
CAPILLARY ELECTROPHORESIS
Definition:-These kind of separations are facilitated by the use of high
voltages, which may generate electro-osmotic and electrophoretic flow of
buffer solutions and ionic species, respectively within the capillary.
Principle:- Capillary electrophoresis is an analytical technique that separates
ions based on their electrophoretic mobility with the use of an applied
voltage. The electrophoretic mobility is dependent upon the charge of the
molecule, the viscosity, and the atom's radius. The rate at which the particle
moves is directly proportional to the applied electric field .
APPLICATION OF ELECTROPHORESIS
1.Estimation of the DNA molecule.
2. Analysis of PCR product.
3. Separation of restricted genomic DNA and RNA.
4. Conformation of newly isolated DNA .
5. Separation of most small fragments of DNA.
6. In forensic science.
7. In determining molecular wt. of protein.