Electrophoresis is a method for separating charged molecules in a liquid under an electric field, utilizing different types of gels like agarose and polyacrylamide for analysis. Two-dimensional gel electrophoresis combines isoelectric focusing and SDS-PAGE to analyze complex protein mixtures, allowing for the resolution of thousands of proteins simultaneously. The technique involves various steps such as separation, staining, and detection, making it highly effective for preparative and analytical purposes.

1. ELECTROPHORESIS
TOPIC:
2D-GEL ELECTROPHORESIS

2. ElectrophoresisElectrophoresis
• Electrophoresis is the migration of charged
molecules, particles or ion in a liquid medium under
the influence of an electric field
• Various types – defined by support used
1. Paper – amino acids, small peptides
2. Polyacrylamide – Proteins, small DNA/RNA (<500bp)
3. Agarose – DNA/RNA
• Good preparative and analytical method

3. PrinciplePrinciple
 Proteins move in the electric field. Their relative
speed depends on the charge, size, and shape of
the protein.

4. Technique ofTechnique of
electrophoresiselectrophoresis
Instrumentation and reagents:
(1) Two buffer boxes contain the buffer used in the
process.
(2) Each buffer box contains an electrode made of either
platinum or carbon, the polarity of which is determined
by the mode of connection to the power supply.
(3)The electrophoresis support on which separation
takes place may contact the buffer directly, or by
means of wicks
(4)The entire apparatus is covered to minimize
evaporation and protect the system
(5) The power supply to provide electrical power.

5. General operations performed inGeneral operations performed in
conventional electrophoresis include:conventional electrophoresis include:
(1) separation(1) separation
(2) staining(2) staining
(3) detection(3) detection
(4) Quantification(4) Quantification
General Procedure

7. The sample
Charge
Size
Shape

8. The electric field
Current
Voltage
Resistance
Heat

9. The Buffer
Composition
Concentration
PH

10. 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 molecules.
Types of Gel:
 Agarose gel.
 Polyacrylamide gel.
GEL ELECTROPHORESIS

11. Gel ElectrophoresisGel Electrophoresis
• Gel electrophoresis uses a cross-linked polymers
(agarose) that contain various pores.
• Pores allow molecular sieving, where molecules e.g.
DNA, can be separated based upon there mobility
through the gel.

12. AGAROSE GELAGAROSE GEL
 A highly purified uncharged polysaccharide
derived from agar.
 Used to separate macromolecules such as nucleic
acids, large proteins and protein complexes.
 It is prepared by dissolving 0.5% agarose in
boiling water and allowing it to cool to 40°C.
 It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds.

13. DNA Gel ElectrophoresisDNA Gel Electrophoresis
 Detection
1. Dye e.g. ethidium bromide
2. Audioradiography 32
P,
3. Blotting
 Uses
1. Analytical- Can determine size of DNA
fragment,
2. Preparative – Can identify a specific fragment
based on size

14. 2D-gel (coomassie stained)2D-gel (coomassie stained)

15. ISOELECTRIC FOCUSINGISOELECTRIC FOCUSING
 Electrophoretic method that separates
proteins according to the iso-electric
points
Is ideal for seperation of amphoteric
substances
Seperation is achieved by applying a
potential difference across a gel that
contain a pH gradient
Isoelectric focusing requires solid support
such as agarose gel and polyacrylamide
gel

16. TWO-DIMENSIONAL ELECTROPHORESISTWO-DIMENSIONAL ELECTROPHORESIS
 This technique combines the technique
IEF (first dimension), which separates
proteins in a mixture according to charge
(PI), with the size separation technique of
SDS-PAGE second dimension).
 The combination of these two technique
to give two-dimension(2-D)PAGE provides
a highly sophisticated analytical method
for analysing protein mixtures.

18. 2D GEL ELECTROPHORESIS2D GEL ELECTROPHORESIS

19.  Using this method one can routinely resolve
between 1000 and 3000 proteins from a cell
or tissue extract and in some cases workers
have reported the separation of between 5000
and 10000 proteins.
 The result of this is a gel with proteins spread
out on its surface. These proteins can then be
detected by a variety of means, but the most
commonly used stains are silver and
coomasie staining.

20. THANK
YOU