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PAGE ELECTROPHORESIS




   PRESENTED BY:
   KOUSHIK DAS
   Roll no.10
   S.I.F, C.U.S.A.T-16
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.
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.
DIFFERENT TYPES OF GEL
1.   Agarose gel
2.   Polyacrylamide gel
3.   SDS (sodium dodecyl sulphate)
4.   Native PAGE
5.   Gradient gel
AGAROSE GEL
Agarose  is one of the component of
agar obtained from certain seaweeds.

Agarose   is usually used at
concentration between 1% and 3%.
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.
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.
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.
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”
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.
PAGE
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.
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.
SDS- PAGE
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
NATIVE -PAGE
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
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
USE OF
ELECTROPHORESIS
Molecular     Biology
Medicines
Quality control
Purity tests
    Fluorescence checks
    Phenol tests
    KOH for white vs. red
Forensics lab.
Genetics
Koushik page electrophoresis

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Koushik page electrophoresis

  • 1. PAGE ELECTROPHORESIS PRESENTED BY: KOUSHIK DAS Roll no.10 S.I.F, C.U.S.A.T-16
  • 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.
  • 11. PAGE
  • 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