Electrophoresis

Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field of electrical charge. It is most often used in life sciences to separate protein molecules or DNA and can be achieved through several different procedures depending on the type and size of the molecules. The procedures differ in some ways but all need a source for the electrical charge, a support medium and a buffer solution. Electrophoresis is used in laboratories for the separation of molecules based on size, density and purity.An electric field is applied to molecules and as they are electrically charged themselves it results in a force acting upon them. The greater the charge of the molecule the greater the force applied by the electrical field and therefore the further through the support medium the molecule will move relative to its mass.

Some example applications of electrophoresis include DNA and RNA analysis as well as protein electrophoresis which is a medical procedure used to analyse and separate the molecules found in a fluid sample (most commonly blood and urine samples).Different types of gels are usually used as the support medium for electrophoresis and this may be in slab or tube form depending on which is more beneficial. Gel slabs enable many samples to be run simultaneously and so are frequently used in laboratories. However, tube gels give a better resolution of the results so are often chosen for protein electrophoresis.

Agarose gel is commonly used for electrophoresis of DNA. It has a large pore structure allowing larger molecules to move easily but it is not suitable for sequencing smaller molecules.

Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than agarose gel making it more suitable for quantitative analysis. This makes it possible to identify how proteins bind to DNA. It can also be used to develop an understanding of how bacteria is becoming resistant to antibiotics through plasmid analysis.

1. electrophoresis Sakshi nayak Msc. 1 st sem Inst.of biomedical sciences

2. introduction It is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes . It may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charges. It was discovered by Tselius in 1930s.

3. Electrophoretic mobility The velocity (v) of charged molecule in an electric field: v = Eq / F where , F = frictional coefficient, which depends upon the mass and shape of the molecule. E = electric field (V/ cm ) q = the net charge on molecule v = velocity of the molecule.

4. Factors affecting electrophoretic mobility Charge – Higher the charge greater the electrophoretic mobility. Size – Bigger the molecule greater are the frictional and electrostatic forces exerted on it by the medium. Consequently, larger particles have smaller electrophoretic mobility compared to smaller particles. Shape – Rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours. Therefore globular protein move faster than fibrous protein.

5. Types of electrophoresis Moving boundary electrophoresis Zone electrophoresis: a ) Paper Electrophoresis b ) Gel Electrophoresis

6. Moving boundary electrophoresis It was first developed by A.Tiselius in the 1930s. The moving boundary method allows the charged species to migrate in a free moving solution without the supporting medium . INSTRUMENTATION: Consists of a U shaped glass cell of rectangular cross section, with electrodes placed on the one each of the limbs of the cell. Sample solution is introduced at the bottom or through the side arm, and the apparatus is placed in a constant temp. bath at 40* C.

7. Zone electrophoresis It involves the migration of the charged particle on the supporting media. Discrete zones are formed . Paper , Cellulose acetate membrane, Starch Gel, Poly acrylamide. Components separated are distributed into discrete zone on the support media. Supporting media is saturated with buffer solution, small volume of the sample is applied as narrow band.

8. Zone electrophoresis

9. Techniques of zone electrophoresis Paper electrophoresis Gel electrophoresis : a) Agarose gel (AGE) b) Polyacrylamide gel(PAGE)

10. General method of operation Saturation of the medium with the buffer Sample application. Electrophoretic separation. Removal of the supporting media INSTRUMENTATION: Electrophoretic chamber Electrodes Supporting / Stabilizing media. (inert to sample and to any developing reagents)

11. Electrophoresis equipment

12. Paper electrophoresis This technique is usefull for the separation of the small charged molecules such as amino acids and small proteins. A strip of filter paper is moistened with buffer and the ends of the paper are i mmersed in the buffer reservoir containing the electrodes.

13. Paper electrophoresis

14. Paper electrophoresis FILTER PAPER : It is the stabilizing medium. We can use whatman filter paper, cellulose acetate filter paper or chromatography paper. APPARATUS : Power pack, electrophoretic cell that contains electrodes, buffer reservoirs, support for paper, transparent insulating cover . SAMPLE APPLICATION : The sample may be applied as a spot(about 0.5 cm in diameter)or as a uniform streak. ELECTROPHORETIC RUN : The current is switched on after the sample has been applied to the paper and the paper has been equilibrated with the buffer . The types of buffer used depends upon the type of separation . Once removed, the paper is dried in vaccum oven . DETECTION : To identify unknown components in the resolved mixture the electrophoretogram may be compared with another electrophoretogram on which standard components have been electrophoresed under identical conditions

15. Gel electrophoresis It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix. Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules. Types of Gel: Agarose gel (AGE). Polyacrylamide gel (PAGE).

16. Gel electrophoresis Slab gel electrophoresis can have either a horizontal or vertical format . Sample is introduced into the wells at the top of the gel.

17. horizontal/ submarin -e gel electrophoresis VERTICAL GEL ELECTROPHORESIS

18. Casting of the gel

19. Gel casting trays 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.

20. Loading of the gel Carefully place the pipette tip over a well and gently expel the sample .The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

21. Running of the gel

22. Staining

23. Agarose gel It is a disaccharide consisting of galactose and 3,4 - anhydrogalactose. A highly purified uncharged polysaccharide derived from agar . Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C.

24. Agarose gel ADVANATGES: 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 . Sharp zones are obtained due to less adsorption. Recovery of protein is good, good method for preparative purpose

25. Polyacrylamide gel It is prepared by polymerizing acryl amide monomers in the presence of methylene- bis -acrylamide to cross link the monomers. Structure of acrylamide (CH2=CH-CO-NH2 ). 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 called Molecular sieving

26. PAGE ADVANTAGES: Gels are stable over wide range of pH and temperature . Gels of different pore size can be formed . Simple and separation speed is good comparatively. Types : Native page Denatured or SDS page

27. Native page( isoelectrophoresis ) No denaturing agents. Proteins separated based on size, charge and shape . It is used when we want to keep the protein active to study conformation , binding of other proteins . VISUALIZATION : Ethidium bromide . Silver or coomasive blue dye .

28. Non-native (sds page) It is negatively charged detergent (sodium dodecylsulfate ). It is used to denature and linearize the proteins . It coated the proteins with negative charge. It is useful for monitoring protein purification as separation based on the size of the particles.

29. Staining of dna To make DNA fragments visible after electrophoresis , the DNA must be stained . The favorite : E thidium bromide . When bound to DNA it fluoresces under UV light(reddish –orange color). Sensitive – detects 0.1ug of DNA. Other alternatives for ethidium bromide : Syber safe Xylene cyanol

30. 2d- gel electrophoresis In the first dimension, proteins are resolved in according to their isoelectric points (PI) using immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis. ( Horizontal separation). In the second dimension, proteins are separated according to their approximate molecular weight using SDS-PAGE. (Vertical separation ).

31. Capillary electrophoresis It is the technique of performing electrophoresis in buffer filled, narrow-bore capillaries, normally from 25 to 100 mm in internal diameter (ID ). A high voltage (typically 10-30 kV) is applied . Capillaries are typically of 50 μm inner diameter and 0.5 to 1 m in length . Due to electroosmotic flow, all sample components migrate towards the negative electrode . The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution , greater sensitivity, and online detection . The capillary is filled with electrolyte solution which conducts current through the inside of the capillary . The ends of the capillary are dipped into reservoirs filled with the electrolyte . Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the electrical circuit.

32. instrumentation A buffer filled fused silica capillary. Two electrode High voltage supply (5-30Kv) Sample injector Detector Buffer solutions

33. Capillary electrophoresis Detectors : Detectors similar to those used in GC, HPLC can be used. Majority of instruments have UV- visible detectors. The mass spectrophotometers are frequently used to give structural information . Alternative detectors modes include fluorescence , laser induced fluorescence .

34. Capillary electrophoresis ADVANATGES : Easy and predictable selectivity. Small sample required Fast separation. Easily coupled to MS DISADVANTAGES : Cannot do preparative scale separations.

35. Pulsed field gel electrophoresis It is a technique used for the separation of large molecules of DNA by applying to a gel matrix in an electric filed that periodically changes direction . The voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA . This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel.

36. Applications DNA Sequencing Medical Research Protein research/purification Agricultural testing Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids , insulin. In food industry It is employed in biochemical and clinical fields i.e. in the study of protein mixtures such as blood serum , hemoglobin and in the study of antigen- antibody interactions. Electrophoresis in combination with autoradiography is used to study the binding of iron to serum proteins . U sed for analysis of terpenoids , steroids and antibiotics. For testing purity of thyroid hormones by zone electrophoresis. Paper chromato -electrophoresis is used to separate free Insulin from plasma proteins. It is used for diagnosis of various diseases of kidney , liver and CVS. It is also used for separation of Scopolamine and Ephedrine using buffer at PH 4.2. Electrophoresis is also used for separation of carbohydrates and vitamins.

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