Gel electrophoresis

This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.

1. Electrophoresis

2. 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.

3. 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 .

4. Factors affecting Electrophoresis Inherent Factors: Magnitude of its charge Charge density Molecular weight External Environmental Factors: Solution Ph Electric Field Solution Viscosity Temperature

5. Electrophoretic Chamber

6. 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.

7. 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.

8. Types of Gels: There are 3 types of gels that are use in electrophoresis: Agarose: For separating larger nucleic acids Polyacrylamide gel: For separating smaller nucleic acids. SDS-PAGE: For denaturing the proteins.

9. 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.

10. Technique: Sampling Electrophoretic run Staining Detection & Quantification

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. Types of Electrophoresis Zone Electrophoresis Slab gel Electrophoresis Disc Electrophoresis Isoelectric Focusing Electrophoresis 2 Dimensional Electrophoresis Capillary Electrophoresis

17. 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.

18. 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.

19. 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.

20. 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.

21. 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. 1 st Dimension – Charge dependent IEP 2 nd Dimension – Molecular weight dependent electrophoresis It achieves the highest resolving power for the separation of DNA fragments.

22. 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.

23. 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.

24. Molecular biology, Microbiology, Biochemistry. Use in DNA fingerprinting. Separation of serum proteins. Use in antigen-antibody species. Use in food industry