ELECTROPHORESIS

TOPIC:2D-GEL ELECTROPHORESIS

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

PrinciplePrinciple

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

Technique of Technique of electrophoresiselectrophoresisInstrumentation 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.

General operations performed in General 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

The sample

Charge

Size

Shape

The electric field

Current

Voltage

Resistance

Heat

The Buffer

Composition

Concentration

PH

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

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.

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.

DNA Gel ElectrophoresisDNA Gel Electrophoresis

Detection 1. Dye e.g. ethidium bromide2. Audioradiography 32P, 3. Blotting

Uses1. Analytical- Can determine size of DNA

fragment, 2. Preparative – Can identify a specific fragment

based on size

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

ISOELECTRIC FOCUSING ISOELECTRIC 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

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.

2D GEL ELECTROPHORESIS2D GEL ELECTROPHORESIS

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.

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