Gel electrophoresis

Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.

1. A Presentation on the Topic GEL ELECTROPHORESIS (MPL101T) Presented by AVINASH KUMAR 1 st semester M.Pharm DEPARTMENT OF Pharmacology

2. Content Introduction Types of Electrophoresis Gel Electrophoresis Principle of Separation Material required for gel Electrophoresis Types of gel Procedure of gel Electrophoresis Factor affecting separation of gel Electrophoresis Application References 2

3. Introduction Tiselius a Swedish biochemist was the Nobel prize in 1948 . He first developed the electrophoresis technique and discover the complex nature of serum protein. Electro- Electric field , Phoresis - separation of ions. The Process of migration of charged particle through a solution under the influence of external electric field is called Electrophoresis. Electrophoresis of positively charged particles is sometimes called cataphoresis , while electrophoresis of negatively charged particles is sometimes called anaphoresis . Electrophoresis is used in laboratories to saperate biomolecules based on size ( such as DNA,RNA, Protein etc.)

4. Types of Electrophoresis 4

5. Gel Electrophoresis Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins. according to their size. Charged molecules move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!). The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size. 5

6. Principle of Separation 6 According to charge : When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. According to size: The smaller molecules move more swiftly than the larger sized ones, as the can travel through the pores more easily than the later.

7. Materials/equipment required for gel electrophoresis Electrophoresis chamber Gel tank Casting tray Tank cover with electrode Power supply Agarose gel Buffer Staining agent (dye) Comb DNA ladder 7

8. Types of Gel 8

9. Preparation of stock solution Weigh 10 mg ethidium bromide into a sterile tube and dissolve in 10 ml sterile distilled water. • The stock is stored at 4°C. B uffers  in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. The most common being,fornucleic acids Tris /Acetate/EDTA (TAE), Tris /Borate/EDTA(TBE). 9 Preparation of Ethidium bromide(Dye) Preparation of buffer

10. Procedure of Gel electrophoresis ( Agarose gel Electrophoresis): Step1- Preparing DNA Sample The DNA is isolated and pre- processed (e.g. PCR., Enzymatic digestion. Made up in solution with some basic dye, mostly blue, to help visualize the movement of sample through the gel. 10

11. Step2-Preparing the Gel 11 Molecular grade agarose gel powder Combine the agarose powder and buffer solution. The agarose solution is boiled until clear

12. Step3- Gel Casting 12 Gel tray and combs Inserting comb Pouring prepared gel into tray Remove combs after gel is cooled down

13. To be continue….. 13 Place the gel in the electrophoresis chamber

14. Step 4- Loading the sample 14 Carefully place the pipette tip over a well and gently expel the sample. A ladder or standard of known size is also loaded

15. Step 5- placing gel tray in Tank 15 Place the casting tray containg the gel into ELECTROPHORESIS rig. Add enough TAE to the gel electrophoresis rig to cover the gel 3-3mm. Place the cover on electrode. Connect both electrode to power source.

16. Step 6- Running electrophoresis Turning on the power supply setup the electric field. The negatively charged DNA samples will start to migrate though the gel and away from negative electrode towards the positive. 16

17. The ethidium bromide stained gel is then exposed to uv light and a picture is taken. DNA bands are visualized in from each lane corresponding to a chamber well The DNA ladder that was looked is also visualized and the length of the DNA bands can be estimated. 17

18. Step 7-Reading the Gel Image 18

19. Factor affecting separation in gel electrophoresis The charge/mass ratio of the sample dictates its electrophoretic mobility . Charge : Higher the charge, greater the electrophoretic mobility. Size : Size is inversely proportional to electrophoretic mobility. Shape : Globular substances move faster than the fibrous ones. 19 The sample

20. An increase in the potential gradient increases the rate of migration. The medium : The inert medium can exert adsorption or molecular sieving effects on the particle influencng its rate of migration. Adsorption : retention of the component on the surface of supporting medium. Molecular r sieving: media such as agar, polyacrylamide, sephadex have cross linked structures giving rise to pores within the gel beads. 20 The electric field

21. the buffer can affect the electrophoretic mobility by: Ionic strength: increase in ionic strength of buffer means a larger share of current is carried by buffer and smaller proportion by sample, while decrease in ionic strength is vice-versa. pH : pH determines the degree of ionization of organic compounds. Where ionization is inversely proportional to pH. 21 The Buffer

22. Application Separation of   Deoxyribonucleic acid (DNA) . Separation of ribonucleic acid (RNA). Separation of protein molecules . It may be used as preparative technique prior to use of other methods such as mass spectroscopy, cloning, DNA Sequences, Southern Blotting for further characterization. Separation of amino acid . Separation of lipoproteins. Separation of enzyme in blood . Separation of antibiotic drug. 22

23. References Instrumental methods   of chemical analysis. By Dr. B.K.Sharma , Page no. 661-670. Instrumental analysis by William Kemp. http://www.intechopen.com/books/gel- electrophoresisprinciples -and-basics 23

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