Vertical Gel Electrophoresis

In this slide contains introduction, principle, application, advantage and disadvantage of Vertical Gel Electrophoresis
Presented by: Shaik Firdous Banu. (Department of pharmacology),
RIPER, anantapur.

1. Vertical Gel Electrophoresis A Seminar as a part of curricular requirement for M. Pharmacy, I Year - I semester Presented by SHAIK FIRDOUS BANU (20L81S0105) PHARMACOLOGY Under the guidance of Dr. P. Ramalingam M.Pharm, Ph.D. Professor & Director of R& D cell

2. 2 Contents Introduction Vertical gel electrophoresis Principle 2D Polyacrylamide Gel Electrophoresis Applications Advantages and disadvantages References

3. 3 Introduction: Gel electrophoresis is a method for separation and analysis of macromolecules like DNA, RNA and protein or their fragments, based on their size and charge. Based on gel casting technique, classified in to : Horizontal Gel Electrophoresis. Vertical Gel Electrophoresis.

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5. Cross-linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of smaller amount of methylenebisacrylamide. 5 Vertical gel electrophoresis

6. Principle: Any charged molecule or ion migrate when placed in an electric field, the rate of migration depends upon its net charge, size, shape and the applied electric current. Can be represented by following equation: V =E* q f Where, V=velocity of migration of the molecule E=electric field q=net electric charge on the molecule f=frictional coefficient 6

7. Types of PAGE 7 PAGE can be classified according the separation conditions into: Native-PAGE: Native gels are run in non-denaturing conditions, so that the analyte’s natural structure is maintained. Separation is based upon charge, size and shape of macromolecules. Useful for separation and purification of mixture of proteins.

8. SDS PAGE : When Sodium dodecyl sulfate detergent SDS added to PAGE the combined procedure is termed as SDS PAGE. It has strong protein denaturing effect. SDS coats proteins molecules giving all proteins a constant charge mass ratio. Proteins migrate along the gel in order of increasing size or molecular weights. 8

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11. Gradient PAGE Unlike fixed concentration gels, gradient gels are formulated with a range of polyacrylamide concentrations. Where the gradient begins with a lower concentration and ends with a higher concentration. 11

12. Importance of Gradient PAGE A much greater range of protein can be separated than on a fixed –percentage gel. Proteins with similar molecular weight can be resolved. The higher the polyacrylamide gels, the smaller the pore Size in the matrix. Higher concentration gels can separate small sized proteins. While low concentration gels, with larger pore size are better at resolving higher molecular weight proteins. 12

13. Gradient gel preparation It uses two chambers, where one contains the acrylamide solution at the lowest gradient concentration, and the other contain the higher acrylamide Concentration. 13

14. Stacking gel (4%): It concentrate all the proteins in one band, so that they will start migrating in running gel all at the same time. Running gel (12%) : Running gel allows to separate the Proteins based on their molecular weight. 14

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16. Running the gel 16 Take out the gels from the casting frame and clamp them in the gel apparatus. When the plates are secured, place them in cassette and then lock. Place them in the gel running tank. Fill the inner chamber of the tank with buffer. Remove the comb carefully.

17. Microgram quantity of the sample is placed over the top of the gel column. Cathode and anode are kept above and below the Column to impose an electric field through the column. 17

18. There is no external solvent space, all the migratory particles have to pass through the gel pores. Different sample components get separated in to discrete migratory bands along the gel column on the basis of electrophoretic mobility and gel filtration effect. 18

19. Visualization Ethidium bromide or coomassie blue dye may be used for this process. If the analyte molecule fluoresce under the UV light, a photograph can be taken of the gel under UV lighting conditions. 19

20. 2D Polyacrylamide Gel Electrophoresis Principle : Proteins were resolved on a gel using isoelectric focusing(IEF) in first dimension. In second dimension in the Presence of sodium dodecyl sulfate. 20

21. First dimension : Isoelectric Focusing Isoelectric point : It is defined as the PH of a solution at which the net charge of the protein become zero. A protein with positive net charge migrate towards cathode becoming less positively charged until it reaches it Pi. While a protein with a negative net Charge migrate towards anode becoming less negatively charged until it reaches it Pi. 21

22. A protein mixture is loaded at the basic end of the PH Gradient gel. Large proteins will move more Slowly through the gel, but with sufficient time will catch up with Small proteins of equal charge. These IPG Strips placed in SDS Gel. 22

23. 23 Applications of polyacrylamide gel electrophoresis: Used for estimation of molecular weight of protiens and nucleic acids. Determination of subunit structure of protien. Purification of isolated proteins. Monitoring changes of proteins contents in body fluids. Identifying disulfide bonds between protein Quantifying proteins Blotting applications Comparison of polypeptide composition of different samples.

24. 24 Advantages of Polyacrylamide Gel Electrophoresis: Stable chemically cross-linked gel. Greater resolving power. Good for separation of low molecular weight fragments. Pore size of the acrylamide gels can be altered in an easy and controllable fashion by changing the concentration of the two monomers. Disadvantages: Generally more difficult to prepare and handle, involving longer time for preparation than agarose gel. Need new gel for each experiment .

25. Reference: John M. Walker. Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins. Springer. 1994; 32(7): 35-38. Bryan John Smith. SDS Polyacrylamide Gel Electrophoresis of Proteins. Springer. 1994; 32: 23-34. Jaap H. Waterborg, Harry R. Matthews. The Electrophoretic Elution of Proteins from Polyacrylamide Gels. Springer. 1994; 32: 169-175. Paula Meleady. Two –Dimensional Gel Electrophoresis. Springer. 2017; 1664: 3-14. 25

26. THANK YOU 26