Agarose gel electrophoresis by Swapnil Anand.pptx

A compact and concise presentation on Agarose gel electrophoresis

1. Definition And Principle Of Electrophoresis Agarose Gel Electrophoresis Submitted to: Prof. Dinesh Kumar Yadav Prof. Rajiv Kumar Yadav Presented by: Swapnil Anand M.Sc 3 rd Sem Department of Botany

2. Electrophoresis A molecule with a net charge will move in a electric field. This phenomenon is termed as electrophoresis, offers a powerful means of separating proteins and other macromolecules, such as DNA and RNA. 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.

3. Principle Of Electrophoresis When a potential difference is applied across an electrode (V) it generates a potential gradient (E). When this potential gradient E is applied, the force on a molecule bearing a charge of q coulombs is F=Eq When a charged molecule move it also experiences a frictional force (f) that retards the mobile molecule. The frictional force depend on strength of electrical field, net charge, size and shape of molecule, pore size of matrix. Thus velocity (v) of charged molecule in electric field is v=Eq/f v/E= q/f μ = q/f Where μ is electrophoretic mobility.

4. Components of Electrophoresis Electrical Current – the flow of electric charge Positive Electrode – the wire that collects electrons Negative Electrode – the wire that emits electrons Porous – containing pores, permeable to fluids and small particles Sieve – a mesh device to filter small particles out of a mixture of larger particles.

5. Charged molecules are separated based on their electrical charge and size . Separation of a Mixture of Charged Molecules Charge Separation Size Separation Analyze Identify Purify Mixture of Charged Molecules Positive Molecules Negative Molecules

6. How Separation Occurs Electrical Charge: Many molecules (amino acids, peptides, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge. When introduced to an electrical current, negatively charged molecules are attracted to the positive electrode and positively charged molecules are attracted to the negative electrode. - + - + - + - + - + - + - - + - - - - Negatively Charged Protein - + - + + Positively Charged Peptide + N N O + Positively Charged Amino Acid

7. Molecule Size: The porous material is made of microscopic particles suspended in a gel. The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules. Porous Material Proteins Entering Porous Material Smallest Move Fastest How Separation Occurs

8. Equipments The equipment required for electrophoresis consists basically of two items, a power pack which supplies current to Electrophoretic unit. The Electrophoresis units are available for running either vertical or horizontal gel systems.

9. Horizontal Gel System Horizontal Gel Electrophoresis is a gel electrophoresis technique in which the gel is present in a horizontal orientation . Horizontal gel electrophoresis is more often used for the separation of DNA and RNA mixtures . Vertical gel system Vertical Gel Electrophoresis is a gel electrophoresis technique in which the gel is oriented vertically. Vertical gel electrophoresis is used to separate mixtures of protein.

10. Horizontal Gel System Vertical Gel System

11. Electrophoretic Unit Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the Gel is being cast, then removed prior to electrophoresis. Sample combs, around which molten agarose is poured to form sample wells in the gel.

12. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Fluorescent dyes to allow visual monitoring of the process of electrophoresis for example ethidium bromide is used to stain nucleic acids, commasive brilliant blue and silver stain are used to detect proteins. Support Media- The support medium cuts down convection currents and diffusion so that the separated components remain as sharp zones. Polyacrylamide gels - Electrophoresis in acrylamide gels is frequently referred to as PAGE, being an abbreviation for polyacrylamide gel electrophoresis.

13. SDS PAGE- SDS– Polyacrylamide gel electrophoresis (SDS–PAGE) is the most widely used method for analysing protein mixtures qualitatively. It is particularly useful for monitoring protein purification and, because the method is based on the separation of proteins according to size, it can also be used to determine the relative molecular mass of proteins. Smallest protein travel farthest distance.

14. Agarose Gel Electrophoresis Agarose is a linear polysaccharide (average relative molecular mass about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose.

15. Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel. The pore size in the gel is controlled by the initial concentration of agarose; large pore sizes are formed from low concentrations and smaller pore sizes are formed from the higher concentrations. Agarose gels are used for the electrophoresis of both proteins and nucleic acids.

16. Agarose Gel Electrophoresis Of DNA Agarose Gel Gel Box

17. Agarose Gel Electrophoresis Of DNA For the majority of DNA samples, electrophoretic separation is carried out in agarose gels. Since the charge per unit length (owing to the phosphate groups) in any given fragment of DNA is the same, all DNA samples should move towards the anode with the same mobility under an applied electrical field. However, separation in agarose gels is achieved because of resistance to their movement caused by the gel matrix.

18. DNA gels are invariably run as horizontal, submarine or submerged gels; so named because such a gel is totally immersed in buffer. Molten Agarose is poured onto a glass or plastic plate, surrounded by a wall of adhesive tape or a plastic frame to provide a gel about 3mm in depth. Loading wells are formed by placing a plastic well-forming template or comb in the poured gel solution, and removing this comb once the gel has set.

19. The gel is placed in the electrophoresis tank, covered with buffer, and samples loaded by directly injecting the sample into the wells. Samples are prepared by dissolving them in a buffer solution that contains sucrose, glycerol or Ficoll, which makes the solution dense and allows it to sink to the bottom of the well. A dye such as bromophenol blue is also included in the sample solvent; it makes it easier to see the sample that is being loaded .

20. All the molecules in the well pile up against the gel within a few minutes of the current being turned on, forming a tight band at the start of the run. Once the system has been run, the DNA in the gel needs to be stained and visualised. The reagent most widely used is the fluorescent dye ethidium bromide. The gel is rinsed gently in a solution of ethidium bromide (0.5 mg cm3) and then viewed under ultraviolet light (300nm wavelength). The ethidium bromide concentration builds up at the site of the DNA bands and under ultraviolet light the DNA bands fluoresce orange-red. Gel Stained with Ethidium Bromide

21. Agarose Gel Electrophoresis Of RNA Like that of DNA, electrophoresis of RNA is usually carried out in agarose gels, and the principle of the separation, based on size, is the same. However, if the study objective is to determine RNA size by gel electrophoresis, then full denaturation of the RNA is needed to prevent hydrogen bond formation within or even between polynucleotides that will otherwise affect the electrophoretic mobility.

22. There are three denaturing agents (formaldehyde, glyoxal and methylmercuric hydroxide) that are compatible with both RNA and agarose. Each of these agents forms adducts with the amino groups of guanine and uracil, thereby preventing hydrogen bond reformation at room temperature during electrophoresis. Denatured RNA stains only very weakly with ethidium bromide, so acridine orange is commonly used to visualise RNA on denaturing gels.

23. Agarose gel electrophoresis is some times used for separation of proteins of high molecular weight but it is not preferred method for separation of small molecular weight protein because the pores of the gel are too big to have a good separation.

24. Application of Agarose Gel Electrophoresis Pore size can be manipulated to increase or decrease molecular sieving. A large range of sizes of nucleic acid can be separated . Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting. Separation of DNA fragments for extraction and purification. It is used in restriction digestion based studies such as restriction mapping, genomic mapping.

25. Selected references Lehninger – Principles Of Biochemistry Wilson K. and Walker J . - Principles and Techniques of Biochemistry and Molecular Biology. Pathfinder Publications – Tools and Techniques