Horizontal Electrophoresis.

In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis.
Presented by: A.Geethanjali (Department of pharmacology),
RIPER, anantapur.

1. 1 A Seminar as a part of curricular requirement for I year M. Pharm I semester Presented by A.Geetanjali (Reg. No. 20L81S0106) Dept. Of Pharmacology Under the guidance/Mentorship of Mr. A.Sudheer ,M.Pharm Associate Professor Dept. of Pharmacology Horizontal electrophoresis

2. 2 Introduction Principle Materials required for run a gel Method for electrophoresis Factors affecting electrophoresis Applications Disadvantages References Contents

3. 3 Electrophoresis is a process of migration of charged particle through a solution under the influence of external electric field. The term electrophoresis was coined from a Greek word “phoresis” which means “Being Carried Away”. The word electrophoresis means “to carry with electricity”. Shorter molecules move faster and migrate faster than longer ones. Electrophoresis is used extensively in DNA, RNA and protein analysis. Introduction

4. 4 Separate molecules from each other on the basis of size and/or charge and/or shape Basis of separation depends on how the sample and gel are prepared. Principle

5. 5 Charge S e p arat i on Size S e p arat i on Negative M o l e c u l e s Positive Molecules Mixture of Charged Molecules

6. 6 Electrophoresis apparatus Agarose gel Gel casting tray Buffer Staining agent(dye) Comb DNA ladder Sample to be seperate Materials required for run a gel

7. 7 Electrophoresis apparatus

8. 8 Agarose gel Agar is a mixture of polysaccharide extracted from sea weeds. Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is Non-toxic. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution.

9. 9 Melt the agarose.it is done by heating in a microwave/ Bunsen flame. after that agarose will be completely dissolved. The pores of an agarose gel are large, agarose is used to separate macro Molecule such as nucleic acids, large protein complex Add ethidium bromide (EtBr) to a concentration of 0.5 μg /ml. Alternatively N ote: EtBr is carcinogen % Agarose (W/V ) Size range ( Kb pairs)for optimals separation 0.5 2-25 0.75 0.7-10 1.0 0.5-10 1.5 0.2-3 2.0 0.1-2 3.0 0.07-1.5 4.0 0.04-0.9 5.0 0.03-0.6 6.0 0.01-0.4

10. 10 Gel casting tray 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 Pour in the melted agarose and insert the gel comb, making sure that no bubbles are trapped underneath the combs and all bubbles on the surface of the agarose are removed before the gel sets.

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12. 12 Buffer

13. 13 Staining agent Ethidium bromide the standard concentration used in staining DNA in gels is 0.5-1ug/mL Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels.

14. 14 EtBr is having carcinogenic activity. Protect from sunlight. Preparation of Ethidium bromide stock solution : Weigh 10mg ethidium bromide into a sterile tube and dissolve in 10ml of sterile distilled water. Other alternatives for ethidium bromide : Methylene blue Syber safe xylene cyanol bromphenol blue

15. 15 Comb : The white/red plastic electrophoresis comb has many tines.  Electrophoresis combs are  used to create the wells in gels for electrophoresis, It is often used to analyze DNA fragments. When a gel is poured, a comb is inserted .

16. 16 DNA ladder: It is a solution of DNA molecules of different length. DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a “Ladder".

17. 17 Voltage: The speed of movement through the gel is determined by the voltage gradient, i.e. the voltage between the electrodes.   voltage,  rate of migration Horizontal gel tanks are generally run at between 5 – 10 V / cm so if your tank has an electrode distance of 10 cm, you would run the gel at 50 – 100V. An ampholyte become positively charged in acidic condition and migrate to cathode,in alkaline condition they become negatively charge and migrate to anode

18. 18 Sample to be separate: DNA samples should be prepared in a volume that will not overflow the gel wells. Samples are typically loaded into the wells with a micropipette. Care should be taken to prevent mixing of the samples between wells.

19. 19 Method for electrophoresis

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21. 21 The rate of migration of solute in an electric field depends on the following factors: Net charge on the particle Mass and shape of the particles pH of the medium strength of electric field Properties of supporting medium Temperature Factors affecting electrophoresis

22. 22 DNA sequencing For the estimation of size of DNA molecule. Agarose gels allow purification of DNA fragments Protein research /purification(separate different types of protein mixtures as well as nucleic acids). It is employed in biochemical and clinical fields i.e , in study of protein mixture such as blood serum, haemoglobins and in the study of antigen-antibody interactions. Applications

23. 23 Electro osmosis is high. Resolution is less compared to polyacrylamide gels. Different sources and batchs of agar tend to give different results and purification is often necessary. Disadvantages

24. 24 Pei Yun Lee, 1   John costumbrado . Agarose Gel Electrophoresis for the Separation of DNA Fragments . Journal of visualized experiments .   2012; 6(2): 2-5 . Jialiang Li, Yushi Yang, Zhou Mao. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide.Springers.2016; 404: 1-5. Smith, S.B., Aldridge, P.K., & Callis, J.B. Observation of individual DNA molecules undergoing gel electrophoresis. PubMed. 1989; 243: 203-206. Voytas . D.  Agarose Gel Electrophoresis. Current Protocols in Molecular Biology. 2001; 23(9): 1-8. References

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