2. What is Electrophoresis ?
Electrophoresis is an analytical
method frequently used in
molecular biology and medicine. It
is applied for the separation and
characterization of proteins, nucleic
acids and subcellular-sized
particles like viruses and small
organelles.
3. GEL MATERIALS- USED IN
ELECTROPHORESIS
At first stage it is done in the free
solution called “Boundary Technique”.
Filter paper, cellulose acetate strips etc.
are used. But that Gel system is
introduced for electrophoresis.
Generally starch gel system was used,
replaced by agarose or polyacrylamide
gels.
4. DIFFERENT TYPES OF GEL
1. Agarose gel
2. Polyacrylamide gel
3. SDS (sodium dodecyl sulphate)
4. Native PAGE
5. Gradient gel
5. AGAROSE GEL
Agarose is one of the component of
agar obtained from certain seaweeds.
Agarose is usually used at
concentration between 1% and 3%.
6. Preparation of Agarose Gel
Agarose gels- formed by suspending dry agarose in
aqueous buffer, then boiling the mixture until a clear
solution is formed.
This is poured and allowed to cool in room temperature
to form a gel.
The pore size is controlled by the concentration of
agarose.
Although essentially free of charges, that are alternating
sugar residues get substituted with carboxyl, methoxyl,
pyruvate or sulphate to varying degrees.
Therefore agarose is sold in different purity grades
based on the sulphate concentration.
7. Uses of Agarose Gel
Agarose are used for electrophoresis
separation of both proteins and nucleic acid.
The pore size of 1% agarose gel are large
relatively to the size of proteins.
Hence, agarose gels are used in
immunoelectrophoresis or isoelectricfocussing.
Availability of low melting temperature
agarose(62-65ᵒC) allows these gel to be
reliquified by heating to 65ᵒC and thus DNA
samples may be recovered.
8. POLYACRYLAMIDE GELS
Preparation:
Cross-linked polyacrylamide gels are
formed from the polymerization of
acrylamide monomer in the presence of
small amount of N,N- methylene
bisacrylamide.
Bisacrylamide is essentially two
acrylamide molecules liked by a mehylene
group used as cross-linked agent.
9. Preparation (continued):
Acrylamide molecules is polymerized in a
head to tail and bisacrylamide molecule.
Polymerization of acrylamide through free
radical catalysis and is initiated by the
addition of ammonium persulphate and the
base N,N,N,N- tetramethylamide (catalyst)
Polymerization of acrylamide can be
photopolymerization where ammonium
persulphate and TEMED are replaced by
“Riboflavin”
10. Preparation (continues):
The gel is placed under bright light for 2-3
h.
Photodecomposition of riboflavin generate
free radicals that initiate polymerization.
Total percentage of acrylamide usually 3%
to 30%
Pore size can varied by changing the
concentration of both acrylamide and
bisacrylamide.
12. SDS-PAGE(Sodium dodecyl sulphate)
For DNA stacking SDS- polyacrylamide
gels is used.
Gels between 10% and 20% are used for
SDS- gel electrophoresis where the small
pore size act as a sieve and separate the
proteins according to their molecular
weight.
Gels are formed in two forms.
Tube gels and cylindrical rods of
polyacrylamide.
13. Uses of SDS-PAGE
SDS is an anionic detergent which
binds to and denature most protein
1.4 gm of SDS binds per gm of
proteins
SDS denature proteins a rod shape
The length of which depends on the
mass of proteins
SDS denature the proteins migrate
through the gel according to there
mass.
15. NATIVE -PAGE
In native page protein are not denatured
and electrophoresis is carried out in a
variety of buffer system
Depending on isoelectric point of protein
and its various pH
Protein separate according to their
mobility and sieving effect of gel
Its aim to detect a particular protein
17. GRADIENT GELS
This is a polyacrylamide gel system
But in gradient gel system is not uniform
pore
The acrylamide concentration varies from
5% at the top to 25% at the bottom of the
gel
Therefore as the sample moves down ,the
pore size decreases
18. ADVANTAGES
1. Protein with much greater range of
molecular weight are separated very
easily
2. In a complex mixture , very low molecular
weight protein travel freely through the
gel
3. Large protein separate immediately due
to the sieving effects of gel
4. Two protein of similar size but slightly
different molecular weights will separate
19. USE OF
ELECTROPHORESIS
Molecular Biology
Medicines
Quality control
Purity tests
Fluorescence checks
Phenol tests
KOH for white vs. red
Forensics lab.
Genetics