Electrophoresis and it's working principles

Electrophoresis introduction and it's principles it's types and conclusion

1. It is the most common process used for the separation of DNA or protein  molecules . It can be attained through variegated procedures depending on the size and type of the molecules. A  support medium ,  buffer solution , and a  source for the  electric charge  are needed to perform electrophoresis. Electrophoresis is very useful in laboratory practices for the bifurcation of molecules based on density, size, and purity. It is used in protein electrophoresis as well as  plasmids , DNA, nucleic acids, and RNA analysis .

2. Applications Of Electrophoresis: DNA analysis Antib iotic testing Vaccine testing Analysis of proteins and antibodies

3. Principle of electrophoresis The fundamental principle of electrophoresis is the existence of charge separation between the surface of a particle and the fluid immediately surrounding it. An applied electric field acts on the resulting charge density, causing the particle to migrate and the fluid around the particle to flow. It is the process of separation or purification of protein molecules, DNA, or RNA that differ in charge, size, and conformation. The charged molecules are placed at one end of the field according to their charge, and an electric field is applied.

4. The charged molecules are placed at one end of the field according to their charge, and an electric field is applied.  On passing electric current, depending upon the kind of charge the molecules carry, they move towards the opposite electrodes – either cathode (negative electrode) or anode (positive electrode). The size, shape, and charge of the molecule remains constant during electrophoresis and determines ionic particle mobility. An ampholyte acts as a positively charged particle or ion migrating towards the cathode in acidic conditions. In contrast, it acts as a negatively charged particle or ion in an alkaline condition and moves towards the anode.

5. Factors affecting electrophoresis Inherent factors Magnitude of the charge of a molecule Charge density of the molecule Molecular weight Its shape, i.e., whether it’s tertiary or quaternary structured

6. External environment The pH of the solution Electric field The viscosity of the solution or agarose concentration The temperature of operation

7. Types of electrophoresis There are mainly two types of electrophoresis:  Zone electrophoresis and moving boundary electrophoresis. These two are further classified into –  Zone electrophoresis : Paper electrophoresis Gel electrophoresis Thin layer electrophoresis Cellulose acetate electrophoresis

8. Moving boundary electrophoresis : Capillary electrophoresis Isotachophoresis Isoelectric Focusing Immuno -electrophoresis

9. Capillary Electrophoresis  Capillary Electrophoresis is used in very small capillaries and microfluid channels. In this method, particles migrate through an electrolytic  solution  under the influence of an electric field. It is further subdivided into two types namely: Gel Electrophoresis:  It is one of the most used electrophoresis techniques. Agarose gel electrophoresis, Polyacrylamide gel electrophoresis, and Starch gel electrophoresis are three different types of gel-based electrophoresis.

10. Paper Electrophoresis:  It is a technique used for separating small charged molecules of proteins or  amino acids  from a sample. 

11. Slab Electrophoresis  Slab electrophoresis is the second type of electrophoresis which is used for separating protein molecules by analyzing the samples using a 1D format. Slab electrophoresis is divided into three major types as follows: Zone Electrophoresis:  It is a technique for the separation of proteins and nucleic acids.  Immunoelectrophoresis :  It is a process of a combination of immuno -diffusion and electrophoresis. Isoelectrofocusing :  It is used for separating charged molecules like proteins and   peptides.

12. hat is Gel Electrophoresis?   [Click Here for Sample Questions] Gel electrophoresis is a type of electrophoresis technique used for separating charged molecules, such as DNA, based on their size. The charged molecules travel when an electric current is passed through a gel. When an electric current is passed through the gel, it creates a positive charge on one end and a negative charge on the other. These charged molecules can be separated by an electric field. DNA molecules migrate from the negative to a positively charged  electrode  in gel electrophoresis.

13. Gel Electrophoresis Procedure   [Click Here for Previous Years' Questions] The process of Gel Electrophoresis is described as follows:  Separate the DNA by adding a blue dye and prepare a solution so that it becomes convenient to keep track of the sample’s movement in the gel. An electric field can be generated with the help of a TAE solution during the electrophoresis process. In order to make the solution add 100ml of TAE to 1g of agarose .  On heating the agarose TAE solution, the agarose gets dissolved.  Cool the solution of agarose TAE and allow it to solidify. A gel slab as well as a well is now prepared to be applied in this experiment. Place the solidified gel in the chamber after filling it up with the TAE buffer. Place the gel near the negative electrode. 

14. Use the DNA sample and DNA ladder in order to cover the wells. Attach the positive and negative points to the power supply and chamber and then switch on the power. Migration can be seen in the DNA sample as a result of the generated electric field. The sample carrying the negative charge will move in the direction of the positive point and deflect away from the negative point.  Once the blue-colored DNA sample can be seen migrating in the gel switch off the  power  supply. Keep the gel on the solution prepared from ethidium bromide. Take a picture of the exposed ethidium bromide after staining it under  Ultraviolet light . The DNA bands can be seen to appear in the lane of the respective well along with the DNA ladder. Hence, the DNA band length can be determined.

15. Immunoelectrophoresis   [Click Here for Sample Questions] Immunoelectrophoresis is defined as the process of precipitation in agar under an electric field.  It involves the combination of immuno -diffusion and electrophoresis. The term “ immunoelectrophoresis ” was coined by  Grabar and Williams  in the year 1953. The process of  Immunoelectrophoresis is described as follows:  Prepare the agarose gel on a glass slide. Using the sample template, move the wells to the application zone. Dilute the sample by 2:3 by adding protein to it. Now, a condition of stability of the diluent solution is possible through accurate  chemical reactions . Add a 5 μl sample in a 5 μl pipette carefully.

16. Place the gel in the electrophoresis chamber and place the sample near the cathode side. The process of electrophoresis should be carried out for 20 mins at 100 volts. Take out 20 μl of antiserum in a trough and incubate it for 8- 20 hours at room  temperature . Soak the agarose gel for 10 minutes in saline solution, then dry it and wash it twice. Dry the agarose gel at a temperature below 70°C and stain it with protein stain solution for 3 minutes. Decolorize the gel in a destaining solution for around 5 minutes. Once the gel is dried, the results can be determined. 

17. Electrophoresis Process   [Click Here for Sample Questions] Electrophoresis is used to fractionate protein molecules, DNA, or RNA based on the density, type, size, and electrical charge by application of  electric current  and a gel. The pores present in the gel act as a sieve allowing the large molecules to move slower than the small molecules. The conditions used during the electrophoresis are altered depending on the desired size range to separate the molecules. As the molecules contain an electric charge, thus, when they are subjected to an electric field, a force starts acting upon them. The  force  applied by the electric field increases as the charge in the molecules increases. Therefore, depending on the mass of the molecule it starts to move through the support medium.

18. Conclusion Electrophoresis can be considered as an electrokinetic phenomenon involving an electric field, supporting medium, and a buffer solution. It has many forensic and clinical applications in our daily life. From using traditional methods of only rectangular gel usage to using enhanced resolution techniques, electrophoresis has come a long way since its discovery in 1807 in Russia. Overall, electrophoresis is a separation technique that can single out biomolecules or charged particles of interest based on their mobility in a given electric field. Gel electrophoresis allows scientists to visualise the sizes of DNA segments and aids in the sequencing of lengths of DNA.