Electrophoresis. separation of proteins ppt

analysis of protein depending on charge is called electrophoresis

1. Subject Name: Instrumental Methods of Analysis. Unit Name: Electrophoresis

2. Introduction Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. Electro refers to the energy of electricity and phoresis from the Greek verb phoros , means to carry across. The movement of charged particles through an electrolyte when subjected to an electric field. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, nucleic acids possess ionisable groups and, therefore, at any given pH, exists in solution as electrically charged species either as cations or anions.

3. History The name ‘ Electrophoresis ’ was coined by Mr. Michealis in 1909 to describe the migration of colloid ions in an electric field. In 1945 Martin and Synge defined in their review Ionophoresis as the electromigration of low molecular weight ions in stabilizing media. The first sophisticated electrophoretic apparatus was developed by Tiselius in 1937. He was awarded the 1948 Nobel prize for his work in protein electrophoresis. He developed the "moving boundary," which later would become known as zone electrophoresis, and used it to separate serum proteins in solution.

4. Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. Under the charge of an electric field these charged particles will migrate either to cathode or to anode, depending on the nature of their net charge. Principle

5. The positive charges particles (cations) move to cathode and negatively charged ones (anions) move to anode. An ampholyte , a molecule that is either positively charged or negatively charged , takes on a positive charge (binds protons) in a acidic solution and migrates towards cathode. In a more alkaline solution, the ampholyte is negatively ionised (gives up protons) and migrate towards anode. Principle

6. Principle The rate of migration of an ion in electrical field depend on factors: Net charge of molecule Size and shape of particle Strength of electrical field Properties of supporting medium Temperature of operation In the process of electrophoresis large molecules have more difficulty in moving through the supporting medium (i.e., gel) whereas the smaller medium has more mobility through it.

7. Factors affecting Electrophoretic Mobility Charge – higher the charge greater the mobility. Size – bigger the molecule greater the frictional and electrostatic forces exerted on it by the medium i.e. larger particles have smaller electrophoretic mobility compared to smaller particles Electric field – increase of migration with the increase of voltage gradient. Buffer – dependence of migration on pH of the buffer . Ionic strength – greater the ionic strength of the buffer solution higher proportion of the current hence electrophoretic mobility

8. Electrophoretic Mobility ( μ) Electrophoretic mobility (μ) of an ion is used, which is the ratio of the velocity of the ion to field strength. μ = q/ 6π r η w here: q= Net Charge on the ion r= Ionic Radius of Solute η = Viscosity of Medium It is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoretic medium. Other factors affecting mobility include endosmotic flow and wick flow.

9. Conventional Electrophoresis Instrumentation includes: Two reservoir for the buffer Power supply and Electrodes Separation medium

10. At any given PH, exist in a solution as electrically charged species either as a cation (+) or anion(-). Under the influence of an electric field these charged particles will migrate either to cathode or anode, depending on the nature of their net charged.

11. It is a multifunctional component in electrophoretic process as it: Carries the applied current Establishes the pH at which electrophoresis is performed Determines the electrical charge on the solute Thus they determine: Type of charge on solute. Extent of ionization of solute Electrode towards which the solute will migrate. The buffer ionic strength will determine the thickness of the ionic cloud. Buffer

12. Buffer’s ionic strength influences the: Conductance of the support Thickness of the ionic cloud surrounding a charged molecule Rate of migration Sharpness of electrophoretic zones With increasing buffer concentration , the ionic cloud increases in size , and the molecule becomes hindered in its movement. High ionic strength buffer yields sharper band separations, also produce more joule heat due to increased current levels , an effect that leads to heat labile proteins. Ionic strength of a buffer composed of mono valent ions is equal to its molarity. Buffer

13. Choice of Buffer depends upon the nature of substance to be separated and the electricity is supplied at a constant current and Voltage. The choice of buffer depends on the isoelectric point of the sample being analyzed . They allow a current to be carried through the sample while resisting pH changes in the overall solution. In DNA electrophoresis, buffers like TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are used most commonly. In protein electrophoresis, SDS (sodium dodecyl sulfate ) is commonly used. Buffer

14. Support Media Supporting medium is an matrix in which the protein separation takes place. Various type has been used for the separation either on slab or capillary form. Separation is based on to the charge to mass ratio of protein depending on the pore size of the medium, possibly the molecular size. Following things can be taken as Supporting Media: Paper Cellulose Acetate Gels

15. Support Media

16. Procedure For Electrophoresis

17. Procedure For Electrophoresis

18. Procedure For Electrophoresis

19. Procedure For Electrophoresis The dye will migrate through the gel toward the positive electrode, as will the DNA. Depending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the ions move through the gel. Smaller fragments will move easier so they will be closer to the positive electrode. Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray

20. Procedure For Electrophoresis Visualisation 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. 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 auto radiogram can be recorded of the gel.

21. Procedure For Electrophoresis There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

22. Procedure For Electrophoresis Quantification of separated protein band by Densitometer: Densitometer is a device that measures the degree of darkness in photographic or semi-transparent material.

23. Types of Electrophoresis

24. Zone Electrophoresis It involves the migration of the charged particle on the supporting media. 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. On application of PD at the ends of a strip components migrates at a rate determined by its electrophoretic mobility.

25. Zone Electrophoresis ADVANTAGES: Useful in biochemical investigations. Small quantity of sample can be analysed. Cost is low and easy maintenance. DISADVANTAGES: Unsuitable for accurate mobility and isoelectric point determination. Due to the presence of supporting medium, technical complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced.

26. Zone Electrophoresis General Method Of Operation: Saturation of the medium with the buffer. Sample application. Electrophoretic separation. Removal of the supporting media. Instrumentation: Electrophoretic chamber. Electrodes. Diffusion barriers. Supporting/ Stabilizing media. (inert to sample and to any developing reagents).

27. Paper Electrophoresis The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation. The support medium is a Filter Paper. Paper of good quality should contain at least 95% α cellulose and should have only a very slight adsorption capacity. Filter paper such as Whatman No. 1 and No. 3 mm in strip of 3cm or 5cm wide have been used to good effect. The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed. Frequently used in isolating proteins, amino acids and oligopeptides

28. Principle of Paper Electrophoresis A mixture of ions or ionisable substance is applied on the centre of a paper, previously immersed in a buffer of known ionic strength. This paper is placed across two trays, filled with buffer, into which two electrodes are immersed. When a voltage is applied across these electrodes, the ions or ionisable substance migrate towards anode and cathode, based on their charges

29. The two arrangements of the filter strips are commonly used. The horizontal & vertical arrangements Both the arrangements are equally viable & the choice usually depends upon personal preferences. Separation takes place in 12 to 14hrs. Advantages: It is economical. Easy to use. Paper Electrophoresis

30. Disadvantages: Certain compounds such as proteins, hydrophilic molecules cannot be resolved due to the adsorptive and ionogenic properties of paper which results in tailing and distortion of component bands. Electro osmosis. Applications: Serum analysis for diagnostic purpose is routinely carried about by paper electrophoresis. Muscle proteins, egg white proteins, milk proteins & snake, insect venom analysis done by this technique. Paper Electrophoresis

31. Gel Electrophoresis Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”. Gel is a colloid in a solid form (99% is water). It is important that the support media is electrically neutral.

32. Gel Electrophoresis Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels. A porous gel acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely.

33. Gel Electrophoresis Agar gel is used for separation of different types of protein mixtures as well as nucleic acids Polyacrylamide is most suitable for separation of nucleic acids. It is also frequently used in separating proteins, peptides and amino acids from microgram quantities of mixed samples

34. Gel Electrophoresis is carried out in two methods: Vertical starch gel electrophoresis Horizontal starch gel electrophoresis Gel Electrophoresis

35. Types of Gel Electrophoresis

36. Studies can be carried out in thin layer of silica, keisulguhr , alumina. ADVANTAGES: Less time consuming and good resolution. APPLICATION: Widely used in combined electrophoretic-chromatography studies in two dimensional study of proteins and nucleic acid hydrolysates. Thin Layer Electrophoresis

37. It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper. It gives sharper bands. Provides a good background for staining glycoproteins. ADVANTAGE: No tailing of proteins or hydrophilic materials. Available in wide range of particle size and layer thickness. Give sharp bands and offer good resolution. High voltage can be applied which will enhance the resolution. Cellulose Acetate Electrophoresis

38. DISADVANTAGE: Expensive. Presence of sulphonic and carboxylic residue causes induced electroosmosis during electrophoresis. APPLICATION: Widely used in analysis of clinical and biological protein samples (albumin and globulins). Alternative to paper electrophoresis. Cellulose Acetate Electrophoresis

39. Moving Boundary Electrophoresis Principle: 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.

40. Moving Boundary Electrophoresis Sample solution is introduced at the bottom or through the side arm, and the apparatus is placed in a constant temp. bath at 40 o C. Detection is done by measuring refractive index throughout the solution. (Schlieren optical system).

41. ADVANTAGES: Biologically active fractions can be recovered without the use of denaturing agents. A reference method for measuring electrophoretic mobilities. Minute concentrations of the sample can be detected.(0.05mg/ml by Interferometric optical system). DISADVANTAGES: Costlier. Elaborate optical system are required. APPLICATION: To study homogenecity of a macromolecular system. Analysis of complex biological mixtures. Moving Boundary Electrophoresis

42. Capillary Electrophoresis Capillary electrophoresis is a separation method based on the differential rates of migration of charged species in an applied DC electric field Electrophoresis was first developed by Swedish chemist Arne Tiselius in the 1930‟s(serum proteins). He was awarded the Nobel prize for his work (1948) The speed of movement or migration of solutes in CE is determined by their charge and size ratios. The principle behind electrophoresis is that charged molecules will migrate toward the opposite pole and separate from each other based on physical characteristics. Small highly charged solutes will migrate more quickly then large less charged solutes.

43. Principle of Capillary Electrophoresis Capillary electrophoresis, then, 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.

44. Capillary Electrophoresis 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 on-line 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. Defined volume of analyte is introduced in to the capillary by replacing one buffer reservoir with sample vial. Electrophoretic separation is measured by detector.

45. Antibodies are produced by immune system in response to foreign macromolecules. Each antibody binds specifically to one feature(epitope) on one macromolecule(antigen). This allows the use of antibodies for detection and quantitation of specific proteins in a complex mixture. When Electrical potential is applied to study of antigen-antibody reactions, it is called Immunoelectrophoresis . The antibody are electrophoretically separated and antigens diffuse towards each other resulting in precipitin arcs where antigen antibody complexes form. This technique has been referred to as I mmunoelectrophoresis . Immuno Electrophoresis

46. Antibody is placed in trough cut parallel to the direction of the electrophoresis. Run the electrophoresis as a result, precipitin arcs will be formed due to Ab-Ag complex formation. A fluid containing proteins antigens is placed in a well in a thick layer of buffered agar and an electric current is applied, antigens will be distributed in separate spots along a line passing through the well and parallel to the direction of current flow. Immuno Electrophoresis

47. Method of Immuno Electrophoresis It is usually carried out in 2% agar gel medium. The antigen mixture is applied into a small circular wells cut out of agar and the initial electrophoretic separation is carried out depending on their charge and molecular weight. After the initial separation; the antibody mixture is then introduced into a narrow slot in the gel about 0.5 to 1.0 cm from the separated antigens. During this period , the antigen components diffuse radially outwards, towards the diffusing antibody and precipitation takes place in elliptical arcs as related antigens and antibodies diffuse into one another.

48. Method of Immuno Electrophoresis Advantages: Spreading of bands is minimized due to the application of the applied field and the pH gradient, high resolution can be achieved. Disadvantages: Carrier ampholytes generally are used in relatively high concentration , a high voltage power source ( up to 2000V) is necessary and power is in the vicinity of 2 to 50 W. As a result the electrophoretic matrix must be cooled. Application: Mainly used for separating protein and peptides. Used in clinical, forensic and human genetics laboratories for the separation and identification of serum protein in research in enzymology, membrane biochemistry, microbiology and immunology.

49. All proteins have an isoelectric point pH . When electrophoresis is run in a solution buffered at constant pH , proteins having a net charge will migrate towards the opposite electrode so long as the current flows. The use of pH gradient across the supporting medium causes each protein to migrate to an area of specific pH. The pH of the protein equals the pH of the gradient, thus resulting in sharp well defined protein bands. A procedure to determine the isoelectric point (PI) of proteins thus, a mixture of proteins can be electrophorised through a solution having a stable pH gradient in from the anode to the cathode and a each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing. Iso Electric Focusing

50. Protein migrate into the point where its net charge is zero – isoelectric pH. Protein is positively charged in solutions at pH below its PI and will migrate towards the cathode & Protein is negatively charged in solution at pH above its PI will migrate towards the anode. They will be in the Zwitter ion form with no net charge so the further movement will cease. Ampholytes (amphoteric electrolytes)- low molecular mass (600-900D) ooligomers with aliphatic amino and carboxylic acid groups with a range of isoelectric points. Ampholytes help maintain the pH gradient in the presence of high voltage. Can also use gels with immobilized pH gradients - made of acrylamide derivatives that are covalently linked to ampholytes. Iso Electric Focusing

51. Method of Iso Electric Focusing pH gradient is established in gel by addition of ampholytes which increases the pH from anode to cathode. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel migrates until each reaches its pH equivalent to its PI. Each species of proteins is therby focussed into a narrow band about its PI.

52. Method of Iso Electric Focusing The Anode of the column is connected to a reservoir containing an acidic solution like Phosphoric A cid and Cathode is connected to a reservoir containing alkaline solution like sodium hydroxide. On opening the two reservoir valves the two solutions are allowed to diffuse into the column from their respective ends, setting up a PH gradient between the acidic anode and the alkaline cathode. The valves are then closed and the current is switched on, causing the carrier ampholytes to migrate until they reach the PH regions where they have no net charge. They will then remain stationary at these points.

53. Isotachophoresis The technique of isotachophoresis depends on the development of potential gradient. PRINCIPLE: Based on principle of moving boundary electrophoresis. A leading electrolyte (e.g. chloride) with a higher mobility than the analytes, and a trailing electrolyte (e.g. glycinate) with a lower mobility are used. Solution in which the separation takes place is normally an aqueous medium, which contains sucrose to provide a higher density to the solution.

54. Isotachophoresis Where the separation by Isoelectric focusing depends on the existence of a pH gradient in the system. The technique of Isotachophoresis depends on the development of a potential gradient. Separation of the ionic components of the sample is achieved through stacking them into discrete zones in order of their mobilities, producing very high resolution. In the example shown here three particle classes with different charges are being separated and preconcentrated via electrophoresis. After the separation is concluded all particles move at a constant speed ( Isotachophoresis )

55. We fill one well with slow trailing electrolyte (T) mixed with samples (S1,S2), and the other well with fast leading electrolyte (L). When we apply an electric field, ions electromigrated through a microchannel according to their electrophoretic mobilities . Sample ions overspeed the slow trailing electrolyte, but cannot overspeed the fast leading electrolyte ; consequently, they focus at the interface. Isotachophoresis

56. Sample continues to accumulate. If sample concentration approaches the concentration of the leading electrolyte, samples self-segregate into discrete zones. The analytes are positioned between the electrolytes and, when the voltage is applied, they migrate in order of decreasing mobility. This establishes the potential gradient; from that point on, all the analytes move at the same speed. Individual zones border one another but represent completely separated components with out overlap. Isotachophoresis

57. In isotachophoresis no background electrolyte(buffer) is mixed with the sample, so current flow is carried only by charged sample ions. Once a faster moving component separates completely from a slower moving one, It creates a region of depleted charge between the two that increases the resistance and therefore local voltage in that region. This increased voltage causes the slower component to migrate faster and close the gap, thereby concentrating it and increasing the conductivity of its zone until it matches that of the faster ion. Ultimately all ions migration at the rate of the faster ion in the zones that differ in thickness, depending on their original concentrations. APPLICATION: Isotachophoresis that been used for the separation of proteins as well as inorganic substances. Isotachophoresis

58. Two-dimensional Gel Electrophoresis (2-d 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 seperation ) In the second dimension, proteins are separated according to their approximate molecular weight using SDS-PAGE. (Vertical seperation ).

59. Advantages of CE Simple Automated High efficiency of separation Short analysis time Low sample volume Ease of operation Ability to separate both charged and non-charged molecules Low cost Different mechanisms for selectivity Use aqueous rather organic solvents hence environment friendly Disadvantages of CE Aged, improperly stored blood samples – degradation products Abnormal Hb – use other means of identification Migration of Hb variant close to HbA – underestimation of Hb A & variant + overestimation of HbA2 Sensitivity & resolution limits

60. 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, haemoglobins and in the study of antigen- antibody interactions. Electrophoresis is also used for separation of carbohydrates and vitamins. Applications of Electrophoresis

61. separation of all fractions of cellular entities, antibiotics, RBC, Enzymes etc is possible. Electrophoresis in combination with autoradiography is used to study the binding of iron to serum proteins. Used 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. Applications of Electrophoresis

62. Introduction to chemical analysis, by “ H.K.Kaur ” Pharmaceutical analysis, by “ Robert.D.Braun ” Instrumental methods of chemical analysis, by “ B.K.Sharma ” • www.aesociety.org • http://www.namrata.co • http://www.biotechnologynotes.com M. Younus, Ilmi Kitab Khana , 2011. Organic Spectroscopy And Chromatography Instrumental methods of chemical analysis by B. K. Sharma. Instrumental methods of chemical analysis by Gurdeep R. Chatwal, S.K. Anand. References:

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