Gel electrophoresis

Introduction,Types Advantages & Disadvantage

1. GEL ELECTROPHORESIS Presented By- Ms. Sayali S. Chavan & Mr. Pranesh P. Memane 1 ST Year M. Pharmacy PDEA′S SGRS college of Pharmacy,Saswad Guided By:- Mrs. Jagtap P.N. HOD of Pharmacology PDEA′S SGRS college of Pharmacy,Saswad 1

2. 2 Introduction Electrophoresis is a process of migration of charged particle through a solution under the influence of external electric field. 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 macromolecules based on size. Electrophoresis is used extensively in DNA, RNA and Protein analysis. 2

3. C o ntd … When a potential difference applied between the two electrodes in a colloidal solution, it has been observed that the colloidal particles are carried to either the positive or the negative electrode. In other words, they behave as if they have electric charge present within them with respect to the dispersion medium. These phenomenon is known as Electrophoresis and may be defined as the migration of the colloidal particles through a solution under the influence of electric field. 3 3

4. Mechanism of electrophoresis: 4

5. Types Of Electrophoresis 5

6. Gel Electrophoresis Gel electrophoresis is separation technique which uses the gel as a separating pocket. Molecules are separated in aqueous buffer supported within a polymeric gel matrix. Based on the molecular size of the substance molecular sieving technique is employed to facilitate the separation. Molecular sieving technique is the one in which electrophoretic mobility and migration of solute is purely depend on viscosity and pore size. Hence the molecular weight decide the migration of macromolecules in the system. 6

7. 4 Gel Electrophoresis 7

8. GEL ELECTROPHORESIS Gel Electrophoresis is carried out in two methods : Horizontal Starch gel electrophoresis. Vertical Starch gel electrophoresis. 5 8

9. 1. Horizontal gel Electrophoresis In this technique the gel bed is placed in horizontal position as shown in fig. Both the ends of gel bed are connected with the electrophoresis buffer solution separately. The gel acts as the pocket in which the components with the smaller molecular size are trapped & it become easy to separate some of specific components. When potential difference is applied across the two ends, components of the mixture get separated on the basis of their electrophoretic ability. 9

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11. 2. Vertical Gel Electrophoresis The technique employed here is also as similar as the Horizontal gel electrophoresis technique in case of principle, but the arrangement of the experiment is differing in these case. In these case, the sample is kept in the midpoint of the separation plate which is at 90 degrees with the ground. The separation is aided by the gravity and the efficiency of the separation is enhanced. 11

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14. Gel Types Agarose  Polysaccharide extracted from sea weed.  Gel casted horizontally  Non-toxic.  Separate large molecules  Commonly used for DNA separations .  Staining can be done before or pouring the gel. Polyacrylamide Gel  Cross-linked polymer of acrylamide.  Gel casted vertically.  Potent neuro-toxic.  Separate small molecules.  Used for DNA or protein separations .  Staining can be done after pouring the gel. 14

15. 1. Agarose gel electrophoresis  Commonly used support medium  Less expensive than cellulose acetate  Equally good separation  Agar is a complex acidic polysaccharide containin g monomers of sulphated galactose  Agarose is a sulfate free fraction of Agar  Gel is prepared in buffer and spread over a microscopic slide  A small sample of serum or biological fluid is applied b y cutting in to the gel with a sharp edge  The electrophoretic rum takes about 90 minutes 8 15

16. Agar & Agarose gel Agar is a mixture of poly saccharides extracted from sea weeds Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L-galactose. Agarose dissolves when added to boiling liquid. It remains in a liquid state until the temperature is lowered to about 40° C at which point it gels . 16

17. 1  The pore size may be predetermined by adjusting the concentration of agarose in the gel.  Agarose gels are fragile. They are actually hydrocolloids, and they are held together by the formation of weak hydrogen and hydrophobic bonds.  The pores of an agarose gel are large , agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes. 17

18. ADVANTAGES:  Easy to prepare and small concentration of agar is required.  Resolution is superior to that of filter paper.  Large quantities of proteins can be separated and recovered.  A ds o r p t i on o f n e ga t i v e l y c h a rg e d prot e i n m ol e c ule i s negligible.  I t ad s orbs prot e i ns r e la t i v e l y l e ss wh e n c o m p a r e d t o ot h e r medium.  Sharp zones are obtained due to less adsorption.  R e co v e r y of prot e i n i s go o d, g o od m e t ho d f or pr e par a t i v e purpose. 18

19. 1 2 DISADVANTAGES:  Electro osmosis is high.  Resolution is less compared to polyacrylamide gels.  D if f e r e n t s o u r c e s a n d b a t c h e s o f a g a r t e n d t o g i v e d if f e r e n t results and purification is often necessary. APPLICATION :  Widely used in Immuno electrophoresis.  T o s e p a r a t e d if f e r e n t t y p e s o f p r o t e in m i x t u r e s a s w ell a s nucleic acids. 19

20. Gel Structure of Agarose: 1 3 20

21. 2. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis- acrylamide to cross link the monomers. Structure of acrylamide (CH 2 =CH-CO-NH 2 ) Polyacrylamide gel structure held together by covalent cross-links. Polyacrylamide gels are tougher than agarose gels. It is thermostable, transparent, strong and relatively chemically inert. Gels are uncharged and are prepared in a variety of pore sizes. Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon c 1 a 4 lled Molecular sieving. 21

22. T ypes of PAGE 1 5 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 or purification of mixture of proteins. This was the original mode of electrophoresis . 22

23. DENATURED-PAGE OR SDS-PAGE: Separation is based upon the molecular weight of proteins. The common method for determining MW of proteins. Very useful for checking purity of protein samples. 23

24. PAGE-Procedure  The gel of different pore sizes is cast into a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom.  Microgram quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such a pH so as to change sample components into anions.  The foot of the gel column is made to dip in the same buffer in the bottom reservoir .  Cathode and anode are kept above and below the column to impose an electric field through the column. 24

25. Conti....  Macromolecular anions move towards the anode down t he gel column.  T h e r e i s no e xt erna l s olv e nt s pa c e , al l t h e m i gra t ory particles have to pass through the gel pores.  Rate of migration depends on the charge to mass ratio.  Di ff e r ent s a m pl e c om p one nt s g e t s e p a r a t ed i nt o d i s c r e t e m i gra t ory b a n ds a l o ng t h e g e l c o l um n o n t h e ba s i s of electrophoretic mobility and gel filtration effect. 25

26. Procedure 26

27. V i s u a l i z a t i o n  After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.  Ethidium bromide , silver, or coomassie blue dye may be used for this process.  Other methods may also be used to visualize the separation of the mixture's components on the gel.  If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. 27

28. Types Of PAGE 28

29. Native PAGE This is generally used for the separation of protein mixture. In this method native gels are run in non-denaturing conditions . Thus the analyte for example protein′s structure is unaffected. The charge, size and shape of the macromolecules to be separated forms the basis of separation. 29

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31. SDS PAGE Unlike native PAGE, here the basis for separation is the molecular weight of proteins. It can also be employed as the common method for determining molecular weight of proteins. It can be a very useful tool for checking purity of protein sample. 31

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33. A d v a n t a g e s 33

34. Applications  Used for estimation of molecular weight of protein and nucleic acids.  Determination of subunit structure of proteins.  Purification of isolated proteins.  Monitoring changes of protein content in body fluids  Identifying disulfide bonds between protein  Quantifying proteins  Blotting applications 34

35. STARCH GEL ELECTROPHORESIS  A suspension of granular starch should be boiled in a buffer to give a clear colloidal suspension.  The suspension on cooling sets as a semisolid gel due to intertwining of the branched chains of amylopectin.  In order to avoid swelling and shrinking petroleum jelly is used . 35

36. ADVANTAGES: o High resolving power and sharp zones are obtained. o The components resolved can be recovered in reasonable yield especially proteins. o Can be used for analytical as well as preparative electrophoresis. DISADVANTAGES: o Electro osmotic effect. o Variation in pore size from batch to batch. 36

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