Electrophoresis

basic introduction to electrophoresis

1. ELECTROPHORESIS Dipesh Tamrakar M.Sc. Clinical Biochemistry 1

2. OVERVIEW Introduction History Principle Factors affecting electrophoresis Instrumentation Basic procedures 2

3. introduction Electrophoresis is the movement of charged particles through an electrolyte when subjected to an electric Field at the given pH Iontophoresis refers to the migration of small ions zone electrophoresis is the migration of charged macromolecules in a porous support medium such as paper, cellulose acetate, or agarose gel film. Cations move towards cathode and Anions move towards anode 3

4. By this technique solutes are separated by their different rates of travel through an electric field. In a clinical laboratory, the macromolecules of interest are proteins in serum, urine, cerebrospinal fluid (CSF), and other biologic body fluids and erythrocytes and tissue. particularly in the separations of proteins, peptides and nucleic acids 4

5. HISTORY Father of electrophoresis- ArneTiselius (Sweeden,1902-1971) Nobel prize in 1948 for chemistry “for his research on electrophoresis and adsorption analysis ,especially for his discoveries concerning complex nature of serum protein” Arne Tiselius in the 1930s devised moving boundary method; "Tiselius apparatus" In 1937, Tiselius with support from the Rockefeller Foundation, developed the moving boundary electrophoresis 5

6. In the 1940s and 1950s ,Zone electrophoresis methods became effective which used filter paper or gels as supporting media. By the 1960s , gel electrophoresis methods are introduced which can separate biological molecules based on minute physical and chemical differences. Gel electrophoresis and related techniques became the basis for a wide range of biochemical methods, such as protein fingerprinting, Southern blot, western blotting procedures, DNA sequencing, and many more 6

7. principle Depending on the kind of charge they carry, ionized solutes move toward either the cathode (- ve ) or the anode (+ ve ) in an electrophoresis system. An ampholyte becomes positively charged in a solution more acidic than its isoelectric point (Pi) and migrates towards the cathode. In a more alkaline solution it becomes negatively charged and migrates towards anode. 7

8. Electrophoretic mobility When a potential difference ( voltage ) is applied across the electrodes, it generates a potential gradient (E)= applied voltage/ distance between the electrode When this E is applied, the force on a molecule bearing charge of “ q ” coulombs is “ Eq ” newtons It is that force that drives a charged molecules towards an electrode Electrophoretic mobility ( ) = Velocity of ion (V)/ field strength (E) On applying potential difference, molecules will separate depending upon  8

9. Factors affecting electrophoresis The rate of migration of a solute in an electric field depends on the following factors- Net charge on the particle size and shape of the particles pH of the medium Strength of electric field Chemical and physical properties of supporting medium Electrophoretic temperature 9

10. Factors affecting electrophoresis Electrophoresis velocity depends on- 10

11. Frictional resistance that retards the movement of this charged molecules This frictional force is a measure of hydrodynamic size & shape of the molecule, pore size of medium and viscosity of buffer The velocity ‘V’ of a charged molecule in an electric field is given by V = Eq / f f= frictional coefficient Even molecules with similar charge will begin to separate if they have different molecular size ;due to different frictional forces Incomplete form of electrolysis ; termination of electric field before the molecules reach electrodes The separated samples are then located by staining with an appropriate dye or by autoradiography; if sample is radiolabeled 11

12. The current in the solution between the electrodes is conducted mainly by the buffer ions, a small proportion being conducted by the sample ions Ohm’s law: Resistance = voltage / current The distance migrated by the ions will be proportional to both current & time During electrophoresis the power (Watt-W) generated in the supporting medium is W= I 2 R Most of this power generated is dissipated as heat If voltage is constant & current causes  Resistance current causes  heat so use of stabilized power supply & low current 12

13. Heating of the electrophoretic medium has the following effects: An increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples . The formation of convection currents, which leads to mixing of separated samples . Thermal instability of samples that are rather sensitive to heat. This may include denaturation of proteins (and thus the loss of enzyme activity). A decrease of buffer viscosity , and hence a reduction in the resistance of the medium. 13

14. The phenomenon of ELECTROENDOSMOSIS affects electrophoretic separation It is due to presence of charge groups on the surface of the support medium, Examples: Paper has some carboxyl groups present Agarose contain sulphate groups Surface of glass walls used in capillary electrophoresis contains silano (Si-OH ) groups In fused-silica capillary tube, above pH value of about 3, silanol groups on the silica capillary wall will ionize generating negative charge sites 14

15. These charge will generate electroendomosis The ionized silanol groups create an electrical double layers When voltage is applied, cations in the electrolyte near the capillary wall migrate towards the cathode, pulling electrolyte solution with them This creates a net electrosmotic flow towards the cathode 15

16. instrumentation 16

17. 17

18. 5 components of electrophoresis The driving force (electrical power) The buffer tank  hold the buffer The support medium  separation takes place The Electrodes- made of platinum or carbon  connects the buffer to power supply. The detecting system The electrophoresis support on which separation takes place may contact the buffer directly or by means of wicks The entire apparatus is covered to minimize separation 18

19. 1. Power supplies Power supplies are available commercially The migration rate can be kept constant by using a power supply with constant current. This is true because, as electrophoresis progresses, a decrease in resistance as a result of heat produced also decreases the voltage. 19

20. Electrophoresis is always carried out in an appropriate buffer. It is essential to maintain a constant state of ionization of the molecules being separated. Buffer ions actually have a double purpose in electrophoresis: They carry the applied current They set the pH at which electrophoresis is carried out Choice of buffer depends on the nature of substance to be separated 2. Buffer system 20

21. During electrophoresis, ions cluster around a migrating particle. The higher the ionic concentration , the higher the size of the ionic cloud and the lower the mobility of the particle . Greater ionic strength produces sharper protein-band separation but leads to increased heat production. This may cause denaturation of heat-labile proteins. Consequently, the optimal buffer concentration should be determined for any electrophoretic system. Generally, the most widely used buffers are made of monovalent ions because their ionic strength and molality are equal. 21

22. Considerations: Buffer used in electrophoresis are good culture media for the growth of microorganism, so they should be refrigerated when not in use. In addition cold buffer is preferred in an electrophoretic run because it improves resolution and decreases evaporation from the electrophoretic support. Buffer used in small volume apparatus should be discarded after each run because of pH changes due to water electrolysis. Large volume of buffer used (>100ml) can be mixed from both reservoir and can be reused. 22

23. 3. Support media The pioneering work on protein electrophoresis by Arne Tiselius was performed in free solution. Persists various problems ; adverse effects of diffusion and convection currents, could be minimized by stabilizing the medium. The support medium cuts down convection currents and diffusion so that the separated components remain as sharp zones. Filter paper Starch gel Cellulose acetate Agarose Polyacrylamide gel 23

24. Filter paper Most appropriate support media Frequently used in separating small particles s/a carbohydrate, amino acids and oligopeptides Not used nowadays because the polar nature of cellulose shows absorptive effect causing diffusion of samples and broadening of separated zones 24

25. Starch gel electrophoresis separates proteins on the basis of surface charge and molecular size, as does polyacrylamide gel. The procedure is not widely used because of technical difficulty in preparing the gel. Was the first gel medium to be used for electrophoresis. It sepa rates proteins by both charge to mass ratio and molecular size Here proteins compacted on the surface of the gel before migrating into it, they formed narrow bands with improved resolution. Starch Gel 25

26. Paper electrophoresis use has been replaced by cellulose acetate or agarose gel in clinical laboratories. Cellulose is acetylated to form cellulose acetate by treating it with acetic anhydride. Cellulose acetate, a dry, brittle film composed of about 80% air space, is produced commercially. Cellulose acetate prepared to reduce electroendosmosis is available commercially. Cellulose acetate is also used in isoelectric focusing. Widely used for separation of lipo-proteins, isoenzymes and hemoglobin variants Cellulose Acetate 26

27. widely used supporting medium Linear polysaccharide of repeating unit agarbiose Used as a purified fraction of agar, it is neutral and, therefore, does not produce electroendosmosis usually used at concentrations of between 1% and 3%. The dried gel can be stored indefinitely. requires small amounts of sample (approximately 2 mL); it does not bind protein and, therefore, migration is not affected. Agarose Gel 27

28. The pore size in the gel is controlled by the initial concentration of agarose . (1-3%) Large pore sizes  formed from low concentration smaller pore size  formed from the higher concentration Essentially pure agarose is free from charge and does not produce electroendosmosis Agarose gels are used for the electrophoresis of both proteins and nucleic acids. Separation for most routine serum application requires an electrophoresis time of 20-30 minutes. 28

29. Polyacrylamide gel electrophoresis involves separation of protein on the basis of charge and molecular size. Layers of gel with different pore sizes are used. The gel is prepared before electrophoresis in a tube-shaped electrophoresis cell. Polyacrylamide gel electrophoresis separates serum proteins into 20 or more fractions rather than the usual 5 fractions separated by cellulose acetate or agarose. It is widely used to study individual proteins (e.g., isoenzymes). Polyacrylamide Gel 29

30. method Separation Detection Quantification General procedure for Cellulose Acetate Paper Electrophoresis Set up electrophoresis set, buffer and require stain and destain reagents Soak paper in buffer and blot additional buffer Apply sample & control by applicator & transfer paper into bridge Run in electrophoresis for required time at specified current, voltage or power 30

31. Paper is then removed and stained, destained and cleared as per required time Detection, analysis and quantification using scan densitometer Most commonly used general protein detecting stain is sulphated trimethylamine dye , Coomassie Brillant Blue- mainly for PAGE Dye that doesn’t stain support media: Ponceau S, Procion blue, Amido black Silver stain (100 times more sensitive than CBB & detects protein down to 0.1 ng) PAS stain for glycoprotein; lectins Quantitative analysis by scanning densitometry Recently bench top system UV detection 31

32. 32

33. # Technical and practical Consideration : Sampling: To achieve a proper balance between sensitive measurements and resolution, the amount of serum protein applied to an electrophoretic support must be optimum. Urine specimens require 50-100 fold concentration or extended application time for adequate sensitivity CSF may or may not require concentration, depending on the staining approach used 33

34. Discontinuities in sample application May be caused by: Dirty applicators Uneven absorbance by sample combs Inclusion of an air bubble if sample is pipetted onto the gel. 3. Unequal Migration Rates : Caused by: Dirty electrodes causing uneven application of the electric field Uneven wetting of the wicks Improper placement of gel causing sagging and uneven thickness Storing gels too close to heat sources causing partially and unevenly dried areas 34

35. 4. Distorted, Unusual or atypical Bands: Distortion may be caused by : Bent applicators, Incorporation of an air bubble during sample application, Overapplication or inadequate blotting of the sample. Excessive drying of the support medium. 35

36. 5. Unusual bands: Hemolyzed samples are frequent cause of increased beta globulin or an unusual band between the alpha 2 and beta. 6. Atypical Bands: Irregular, sharp protein zone is seen at the starting point due to denatured protein. # Therefore , control serum should be run with each electrophoresis which helps in evaluation of its quality. 36

37. 37

38. 38

39. 39