Electrophoresis for the students of uG and PG

This topic understands by the students of UG and PG of botany, biotechnology

1. Electrophoresis Khusboo Salame M. Sc. II Semester (Food & Nutrition) Govt. D.B. Girls PG College, Raipur

2. What is Electrophoresis? Electrophoresis is an electrokinetic process that separates charged particles in a fluid using a field of electrical charge. Electrophoresis is used in laboratories for the separation of molecules based on size, density, and purity. In the year 1807, Ferdinand Frederic Reuss was the first person to observe electrophoresis. He was from Moscow State University. Anaphoresis is the electrophoresis of negative charge particles or anions whereas cataphoresis is electrophoresis of positive charge ions or cations.

3. Electrophoresis Principle and its types Charged macromolecules are placed in the electric field and move towards the negative or positive pole based on their charge. Nucleic acid has a negative charge and therefore it migrates towards the anode.

4. Types of Electrophoresis: Capillary electrophoresis Gel electrophoresis Paper electrophoresis Slab electrophoresis Immunoelectrophoresis Isoelectrofocusing

5. What is gel electrophoresis? Gel electrophoresis  is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another.

6. Two Basic Types of Materials are used to make Gels Agarose Polyacrylamide Agarose is a natural colloid extracted from sea weed. Agarose gels can be processed faster than polyacrylamide gels.

7. Visualizing the DNA fragments Once the DNA has migrated far enough across the gel, the electrical current is switched off and the gel is removed from the electrophoresis tank. To visualize the DNA, the gel is stained with a fluorescent dye that binds to the DNA and is placed on an ultraviolet transilluminator which will show up the stained DNA as bright bands.

8. Applications of Gel Electrophoresis DNA can be separated by electrophoresis to: Visualize bands of a molecular marker to genotype individual plants Verify amplification by PCR or sequencing reactions Check the quality and quantity of genomic DNA after DNA extraction Separate DNA fragments to clone a specific band

9. Paper Electrophoresis Low-voltage paper  electrophoresis  is the simplest and cheapest form of electrophoresis. A strip of commercially available  chromatography  paper is soaked in buffer and placed with one end in each buffer reservoir (connecting wicks may be used). It is important that the paper is saturated with the buffer since it is the buffer that conducts the majority of the current. A spot of sample is placed in the center of the strip. 

10. Applications of Paper Electrophoresis A simple, inexpensive, and accurate laboratory procedure for various research and clinical studies. used in separation and identification of alkaloids. used for testing suitability of municipal water supplies, toxicity of water, and other environmental components. Drug-testing industry uses paper electrophoresis to determine presence of illegal or recreational drugs in job applicants and crime suspects

11. Immunoelectrophoresis Immunoelectrophoresis is a general name for a number of biochemical methods for the separation and characterization of proteins based on electrophoresis and reaction with antibodies. It is a process of a combination of immuno-diffusion and electrophoresis. The term “ immunoelectrophoresis ” was first coined by Grabar and Williams in 1953.

12. Principle of Immunoelectrophoresis When an electric current is applied to a slide layered with gel, the antigen mixture placed in wells is separated into individual antigen components according to their charge and size.

13. Applications of Immunoelectrophoresis   Immunoelectrophoresis created a breakthrough in protein identification and in immunology. Used to analyze complex protein mixtures containing different antigens. The medical diagnostic use is of value where certain proteins are suspected of being absent (e.g., hypogammaglobulinemia) or overproduced (e.g., multiple myeloma). This method is useful to monitor antigen and antigen-antibody purity and to identify a single antigen in a mixture of antigens.

14. Isoelectric focusing electrophoresis Isoelectric focusing (IEF), also known as electrofocusing , is a technique for separating different molecules by differences in their isoelectric point ( pI ). It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

15. Principle of Isoelectric Focusing (IEF) Isoelectric Focusing or IEF is a method of separating proteins according to their Isoelectric points in a pH gradient. Isoelectric point denoted as pI is defined as the pH at which protein carries no net charge or pH at which protein becomes immobile in an electric field.

16. Applications To determine the isoelectric point(Pi) of a protein. To separate isoenzymes. To fractionate proteins with higher resolution. To study mono, di and tri substituted derivatives of protein. To separate all amphoteric substances.