Gel electrophoresis complete study

gel electrophoresis is technique which we used to separate or purify some macromolecules like DNA RNA & proteins.

1. Mandira S. Bhosale MSc.sem 1 Gel electrophoresis

2. Electrophoresis is a technique used to separate and sometimes purify macromolecules that differ in size, charge or conformation. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds , allowing them to be separated from one another. The gels are porous and the size of the pores relative to that of the molecule determines whether the molecule will enter the pore and be retarded or will bypass it. Thus separation not only depends on the charge but also on its size . Resolution of a sample is sharper and better in a gel than in any other type of medium. What is gel electrophoresis ?

3. Purpose of gel electrophoresis : A method of separating DNA Can be used to separate different size of . DNA . RNA . PROTEINS To purifying DNA, RNA ,Proteins.

4. Specialized gel electrophoresis techniques : 1 ) Discontinuous (disc) gel electrophoresis 2) Gradient gel electrophoresis 3) High voltage electrophoresis 4) Isoelectric focussing 5) 2 D gel electrophoresis 6) Pulse field gel electrophoresis 7) Immuno electrophoresis 8) Electrophoresis on cellular gels 9) Capillary electrophoresis

5. Principle of Gel electrophoresis : Positive or negative electrical charges are frequently associated with biomolecules. When placed in an electric field, charged biomolecules move towards the electrode of opposite charge due to the phenomenon of electrostatic attraction. The molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths . This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule

6. DNA and RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel . IN case of proteins, first mix it with a detergent called sodium dodecyl sulfate. This treatment makes the proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward the positive end of the gel and be separated.

7. Apparatus and requirements: Electrophoretic apparatus includes casting tray with comb to make wells. The casting tray is made up of glass or plastic. The comb contains varying number of teeth in order to help in formation of well.

8. Support media used: The kind of supporting matrix used depends on type of molecules to be separated and the desired basis for separation: charge, molecular weight or both It is important that the support media is electrically neutral . Presence of charge group may cause:-Migration retardation. 1) Starch – high resolving power , analysis of isoenzyme patterns , many drawbacks . 2) Agar/ Agarose – two galactose based polymers, Agarose , Agaropectin , least sieving action , used in immunoelectrophoresis . 3 ) Polyacrylamide gel – neurotoxic components, N,N’- methylenebisacrylamide ,ammonium persulphate, tetramethylenediamine , pore size can be controlled , mostly used and suitable for proteins and nucleic acid separation.

9. 4 ) Agarose acrylamide gel – used to separate high molecular weight nucleic acids, highly porous and rigid matrix. AGE have fairly large pore sizes and are used for separating larger DNA molecules while PAGE are used to obtain high resolution separations for smaller DNA molecules . The pores present in gels

10. Buffer – Buffers 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, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE).

11. Solubilizers – Class of substances which destabilizes the structure of proteins are known as Solubilizers. provides an easy way to estimate the number of polypeptides in a sample. Urea : disrupts hydrogen bonds . ds DNA rendered into ss DNA. SDS : sodium dodecyl suphate, anionic detergent , disrupts hydrophobic reaction and imparts Negative charge. β - mercaptoethanol : breaks disulfide linkages.

12. DNA LADDER/ STANDARDS:

13. Tracking dye: used for tracking the rate of molecular movement through out experiment. xylene cyanol , bromophenol blue , orange G etc. Detection and quantification: staining by E thidium bromide to detect the molecules under UV light source. Blotting Southern blotting (for DNA) Northern blotting (for RNA) Western blotting (for protein)

14. Electrophoretic procedure:

15. Resolution of a sample is sharper and better. Simple to perform. Inexpensive. Can test DNA from any type of evidence. Gel suppresses thermal convection as application of electric supply. The gel can be altered and can give false results. Trained person should required to minimize manual error. If the gel do not have a uniform pore size, resolution will be not great. advantages disadvantages

16. Applications : To determine paternity. Compare similarities and differences between species. To diagnose genetic diseases. Useful in estimation of size of DNA molecules. Useful in plant breeding and genomics for genotyping. Used in analysis of PCR products specially in molecular genetic diagnosis or genetic fingerprinting . Determination of DNA sequences . Determination of molecular weight of proteins . Useful in RFLP and DNA footprinting .

17. References : https:// www.khanacademy.org/test-prep/mcat/chemical-processes/separations-purifications/v/gel-electrophoresis https:// www.slideshare.net/nasira55/gel-electrophoresis-42910366 https:// www.youtube.com/watch?v=dSFQHt-zIQs https:// www.youtube.com/watch?v=4OJAzQsZnbo Biophysical chemistry ( principles and techniques) by Upadhyay page no. 435

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