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 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 gel matrix.
 What is gel?
Gel is a cross linked polymer whose composition and porosity is chosen based on
the specific weight and porosity of the target molecules.
➢ Types of gels used: Polyacrylamide gel, Agarose gel
types

electrophoresis

1D gel 2D gel
electrophoresis electrophoresis

Isoelectric point
SDS-PAGE Native-PAGE IEF in the 1D and by
mass in 2D
TYPES OF GEL ELECTROPHORESIS

AGAROSE GEL POLYACRYLAMIDE GEL


Poured horizontally Poured vertically
Separate large molecules Separate small molecules
Non toxic Neurotoxin
Mostly used for DNA separation Used for DNA or Protein separation
Staining step: before or after pouring Staining step: after pouring the gel
the gel Ethidium bromide is mostly Coomassie blue stain is mostly used
used
 2DE was first independently introduced by O'Farrell and Klose
 It is abbreviated as 2DE
 Form of gel electrophoresis
 Commonly used to analyze and separate proteins
 Medium: Starch = proteins
Agarose gel= very lager proteins, nucleic acid, nucleoproteins etc.
Acrylamide gel= proteins and nucleic acid
BASIS OF SEPARATION
• Begins with electrophoresis in 1D.
• Separate molecules perpendicularly from first to create an electropherogram
• 1D molecules are separated linearly according to their isoelectric points
• Can be performed in the tubes of smaller diameter
• 2D molecules are separated at 90’ from electropherogram according to
molecular mass in normal SDS-PAGE
• Procedure can be adopted by combining IEF and PAGE
PRINCIPLE
 In 2DGE proteins are separated as per isoelectric point and protein mass,
 Separation of proteins by isoelectric point is called IEF (IEF-Isoelectric
Focusing)
 When a gradient of pH is applied to gel and electric potential is applied, one
end becomes more +ve than the other.
 All pH values other than their isoelectric point, proteins will be charged.
 If they are +vely charged, the will be pulled towards –ve and vice versa.
 In separating proteins by mass, the gel treated with sodium dodecyl sulphate
(SDS) along with other reagents (SDS-PAGE in1D) this denatures the proteins
and binds a no. of SDS molecules roughly proportional to the proteins length.
Because proteins length is roughly proportional to its mass, since the SDS
molecules are –vely charged, the result of this is that all of the proteins will
have applied the same mass-to-charge ratio as each other.
APPLICATIONS
 Forensics
 Molecular biology
 Genetics
 Microbiology
 biochemistry
 2D gel electrophoresis is generally used as component of proteomics.
 The step used for the isolation of proteins for further characterization by
mass spectroscopy.
 In the lab we use this technique for 2 main purposes:
!. For the large scale identification of all proteins in a sample.
2. Differential expression, to compare two or more samples to find differences in
their protein expression.
APPLICATIONS
 DNA can be separated by gel electrophoresis to:
visualize band of molecular marker to genotype individual plant
Verify amplification by PCR or sequencing reaction
Check quality and quantity of genomic DNA after DNA extraction
➢ A slightly different but related technique, known as western blot, involves
separating proteins by gel electrophoresis and probing with labeled antibodies
for specific proteins.
➢ Evidence in criminal cases
➢ To determine paternity.
➢ To diagnose genetic diseases.
➢ to determine kindship in animals.
➢ Compare similarities and differences between species.
➢ Determine genetic kindship among species.