Electrophoresis, principle and types

Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.

In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.

Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.

Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

1. TYPES OF ELECTROPHORESIS By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. contents Introduction History Elecrophoresis Principle Types of electrophoresis Application Conclusion Reference

3. Introduction When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode. In other words , they behave as if they are electrically charged w.r.t . the dispersion medium. This phenomenon is known as electrophoresis. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions. Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

4. History 1939- zone electrophoresis developed. 1950- agar gel electrophoresis 1959- acrylamide gel first used. 1971- SDS electrophoresis. 1979- agarose gel electrophoresis. 1983- capillary electrophoresis.

5. Electrophoresis Electrophoresis is defined as the “migration of charged molecules under the influence of an external electric field” Electrophoresis is a technique used for the separation of biological molecules based on their movement due to the influence of a direct electric current. The technique was pioneered in 1937 by the Swedish chemist Arne Tiselius for the separation of proteins. It has now been extended to the separation of many other different classes of biomolecules including nucleic acids, carbohydrates and amino acids.

6. PRINCIPLE Any charged ion or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge, size, shape and the applied electric current. Can be represented by following eq. E * q v = --------------- f

7. v = velocity of migration of the molecule. E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility ,denoted by µ . where, µ = v/E OR µ = q/f

8. GEL ELECTROPHORESIS 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 a gel matrix. Types of Gel: Agarose gel. Polyacrylamide gel.

9. GEL ELECTROPHORESIS

10. Agarose Gel Electrophoresis A highly purified uncharged polysaccharide derived from agar. Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. The cross linked structure gives the gel good anticonventional properties.

11. POLYACRYLAMIDE GEL ELECTROPHORESIS Commonly used components: Acrylamide monomers, Ammonium persulphate , Tetramethylenediamine (TEMED), N,N’-methylenebisacrylamide . These free radicals activate acrylamide monomers inducing them to react with other acrylamide monomers forming long chains. Used to separate most proteins and small oligonucleotides because of the presence of small pores.

12. Isoelectric focusing When electrophoresis is run in a solution buffered at constant pH , proteins having a net charge will migrate towards the opposite electrode so long as the current flows Electrophoretic method that separates proteins according to the iso -electric points 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

13. Capillary Electrophoresis Capillary electrophoresis, is the technique of performing electrophoresis in buffer-filled, narrow-bore capillaries, normally from 25 to 100 pm in internal diameter . The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the electrical circuit

14. 2D- Electrophoresis Two-dimensional gel electrophoresis is widely used to separate complex mixtures of proteins into many more components than is possible in conventional one-dimensional electrophoresis. Each dimension separates proteins according to different properties.

15. Immuno Electrophoresis It is a biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies . All variants of immunoelectrophoresis require immunoglobulin, also known as antibodies  reacting with the proteins to be separated or characterized. The agarose was chosen as the gel matrix because it has large pores allowing free passage and separation of proteins, but provides an anchor for the immuno precipitates of protein and specific antibodies

16. Application Determination of gene sequence Isolation of entire chromosome. separation and characterization of protein. DNA fingerprinting evidence. Restriction mapping of DNA

17. References Biophysical chemistry- Upadhyay & Nath Instrumental analysis- Skoog and Holler www.Slideshare.com www.authorstream.com