Electrophoresis

Definition• Electrophoresis is a method whereby charged molecules in solution,

chiefly proteins and nucleic acids, migrate in response to an electrical field.

• This technique was firstly developed by the Arne Tiselius in 1930 for the study of serum protein.

• Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size.

Purpose of Electrophoresis:• To determine the number, amount & mobility of components in a given

sample or to separate them.

• To obtain the Information about the electrical double layer surrounding the particles.

• To determine the molecular weight of protein molecules.

Factors affecting Electrophoresis

Inherent Factors:

• Magnitude of its charge

• Charge density

• Molecular weight

External Environmental Factors:

• Solution Ph

• Electric Field

• Solution Viscosity

• Temperature

Electrophoretic Chamber

Principle of Electrophoresis• Positive & negative electrical charges are frequently associated with

biomolecules.

• Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis.

• When they placed in electric field , charged biomolecules move towards the electrodes of opposite charge due to the phenomenon of electrostatic attraction.

• An ampholyte become positively charged in acidic conditions & migrate to cathode, In alkaline conditions they become negatively charge & migrate to anode.

• Can be represented by the following equation:

u=v/E = q/f v = velocity of migration of molecules

E = Electric Field in volts per cm

q = Net electric charge on the molecule

f = frictional coefficient

• Electrophoretic mobility of the molecules is directly proportional to charge density.

• Higher the charge greater the electrophoretic mobility.

Types of Gels:• There are 3 types of gels that are use in electrophoresis:

1. Agarose:

For separating larger nucleic acids

2. Polyacrylamide gel:

For separating smaller nucleic acids.

3. SDS-PAGE:

For denaturing the proteins.

Buffers:• Buffers in gel electrophoresis are used to provide ions that carry a

current & maintain the pH at a relatively constant value.

• The buffer ionic strength will determine the thickness of the ionic cloud.

• Buffers used are made monovalent ions because their valences & molality are equal.

Technique:1. Sampling

2. Electrophoretic run

3. Staining

4. Detection & Quantification

1- Sampling• The sample is allow to fall into the sample wells.

• The sample may applied as a spot about 0.5cm in diameter or as uniform streak.

• The sample is then placed into the electrophoretic chamber in contact with the buffer.

2- Electrophoretic run• The current is switched on after the sample has been applied to the paper

& the paper has been equilibrated with the buffer.

• The types of buffer used depends upon the type of separation.

• It will cause the negatively charged proteins or nucleic acids to migrate across the gel away from the negative electrode.

• Smaller biomolecules travel farther down the gel, while lager ones remain closer to the point of origin.

3- Staining• The sample is then stained & dried after washing out the excess dye.

• Amido Black B or members of Coomassie Brillant Blue series are the commonest dyes.

• The amount of dye taken up is dependent on the type of protein, degree of denaturation & quality of dye.

4- Detection & Quantification:• Detection can be achieved by using UV light.

• DNA may be visualized using ethidium bromide which then intercalated into DNA, fluorescence under UV light

• Proteins may be visualized using silver stain or Coomassie Brillant Blue dye.

Downstream Processing• After separation, An additional separation method may be used.

• The gel will then be physically cut & the protein complexes extracted from each portion separately.

• This can provide a great deal of information about the identities of the proteins in a complex.

Types of Electrophoresis1. Zone Electrophoresis

2. Slab gel Electrophoresis

3. Disc Electrophoresis

4. Isoelectric Focusing Electrophoresis

5. 2 Dimensional Electrophoresis

6. Capillary Electrophoresis

1- Zone Electrophoresis• Produce zone of proteins that are heterogeneous & physically separated

from one another.

• Classified according to type & structure of the support material e.g. AGE, CAE, PAGE etc.

2- Slab Gel Electrophoresis• It is primary method used in clinical chemistry lab.

• It has ability to simultaneously separate several samples in one run.

• It uses a rectangular gel regardless of thickness.

• Gels are cast on sheets of plastic backing.

• It is useful in separation of serum proteins, isoenzymes, lipoproteins, hemoglobin & fragments of DNA & RNA.

3- Disc Electrophoresis• 3 Gel system

1- Small pores separating gel (running gel)

2- Larger pore separating gel (stacking gel)

3- Thin layer of large pore monomer solution (sample gel)

• All proteins migrate easily through the large-pore gels.

• This improves resolution & concentrate protein components at the border.

• Discontinuities in electrophoretic matrix caused by layers of gels.

4- Isoelectric Focusing Electrophoresis • It is separation method that resolves proteins markers on the basis of their

isoelectric points.

• Proteins migrate through a zone in a medium where the pH of the gel matches its PI.

• At this point, the charge of the protein becomes zero & its migration ceases. It becomes focused.

• A high voltage power source is needed because carrier ampholytes are used in relatively high concentrations. Thus it must be cooled.

• It is to test for variant Hb. Also use in clinical laboratories for muscle extract & serum extract.

5- 2-Dimensional Electrophoresis • This technique combines the techniques of IEF which separates proteins

in a mixture according to charge (PI) with the size separation technique of SDS-PAGE.

• 1st Dimension – Charge dependent IEP

• 2nd Dimension – Molecular weight dependent electrophoresis

• It achieves the highest resolving power for the separation of DNA fragments.

6- Capillary Electrophoresis• Separation in narrow bore fused silica capillaries filled with buffer.

• Sample is loaded after filling capillary with buffer & electric field applied.

• Electro-osmotic flow (EOF) controls the amount of time solute remain in the capillary.

• Cations migrate fastest due to EOF & electrophoretic attraction towards the cathodes.

• Anions move slower because EOF is slightly greater than the attraction towards the anode & repulsion from cathode.

Applications• DNA sequences can be isolated, analyzed & cloned.

• Synthesis of new antibiotics.

• Analysis of bacteria in response of antibiotics.

• Purifications, processing, & analysis of vaccines e.g. polio vaccine.

• Protein & DNA analysis.

• Determination of impurities.

• Analysis of carbohydrates & macromolecules.

• Analysis of inorganic anions/ metal ions.

• Molecular biology, Microbiology, Biochemistry.

• Use in DNA fingerprinting.

• Separation of serum proteins.

• Use in antigen-antibody species.

• Use in food industry