Basics of electrophoresis
- 1. Electrophoresis Kalpna Assistant Professor & Head Department of Biotechnology AITH, Kanpur
- 2. Electrophoresis • Content Introduction and Definitions Basics of electrophoresis Theory of electrophoresis Electrophoresis instrumentation Phenomenon of electroendosmosis Electrophoretic technique General procedures Types of Electrophoresis Applications
- 3. Introduction and Definition • 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” • Term electrophoresis describes the migration of charged particle under the influence of an electric field • Many biological molecule such as amino acid, nucleic acid and peptides posses ionisable groups, therefore ,at any given pH, exist in solution either as cations (+) or anions(-)
- 4. Basics of Electrophoresis • “electrophoresis”-a technique for separating molecule on the basis of their charge and size • charged molecule moves to their counter charge electrode but electric field removed before it reaches the electrode • Movement of charged species in an electric field gives differential mobility to the sample molecule based on the charge and consequently resolve them • Movement of the charged particle is retarded with the addition a polymeric gel so that a sufficient time is available for resolving the sample.
- 5. Molecule in an Electric Field . . http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html E Q+ QEf*u
- 6. Theory of Electrophoresis Suppose a charged particle of net charge q , the external electric field is E, then the force F responsible for giving electrophoretic mobility, F= q.E…………………………………………….Eq (1) This force derive the charged moleceule towords an electrode However a frictional resistance reatards the movement of this charged molecule The friction forces (F) which is opposing the movement of the charged particle is as follows- F= ƒ. v……………………………………………Eq (2),(here ƒ-frictional coffecient & v-velocity of the electrophoretic mobility) The velocity of charged molecue (v)=Eq/f………………..Eq(3) E=V/d---------------------Eq(4) (here d-distance between the electrode, V-potential difference between electrode) The movement of a spherical through a liquid medium (gel) of the viscosity ç, the friction coefficient ƒ is given by : f= 6Ðçrv……………………………………………Eq (4) Frictional cofficient(f) depands on- The hydrodynamic size of the molecule The shape of the molecule The pore size of the medium Viscosity of the buffer
- 7. Factors affecting electrophoresis • Electrophoresis velocity depends on- • Magnitude of its charge • Charge density • Molecular weight • Shape Inherent Factors • Solution pH • Electric field • Solution viscosity • Temperature External environment-
- 8. Electrophoretic Mobility, μ • More commonally electrophoretic mobility(μ) of an ion is used , the ratio of the velocity of the ion to field strength • When a potential difference applied, molecules with overall charges begin to separate owing to their different electrophoretic mobilities • Even molecules with similar charges begin to separate if they have different molecular size, since they experience different frictional forces • Defined as the ratio of the particles velocity to the strength of the driving field 𝜇 = 𝑣 𝐸 Therefore, 𝜇 = 𝑄 𝑓 ⇒ 𝑄 = 𝜇𝑓 - Now the velocity depends on the particle properties like shape , size, charge and mass
- 9. Contd…………… • Some forms of electrophoresis rely almost totally on the different charges on molecules to effect separation • Other method exploit differences in molecular sizes and therefore encourage frictional effects to bring about separation • The component separated according to their electrophoretic mobility, provided electric field is removed before the molecule in the sample reach the electrodes • Electrophoresis is thus an incomplete form of electrolysis
- 10. Contd…………. • Separated molecules are then located by staining with an appropriate dye or by autoradiography if sample is radiolabelled • Current between the electrode is conducted largely by buffer ion,a small portion being conducted by the sample ions • According to ohms law- V=IR (where V-applied voltage,I-current,R-resistance) • By increasing applied voltage ,accelerate electrophoretic separation • However this generate one of the major problem for most form of the electrophoresis, namely the generation of heat
- 11. Phenomenon of electroendosmosis • During electrophoresis, the power (W ,watts) generated in supporting medium W=I2R • Most of the generated power dissipated as heat, which causes heating of the electrophoretic medium with following effects- An increased rate of diffusion of sample & buffer ions leading to broading of the separated samples The formation of convection current, which leads to mixing of separated samples
- 12. Contd………. Thermal instability of heat sensitive sample include denaturation of protein or loss of activity of enzymes A decrease of buffer viscosity and hence a reduction in the resistance of the medium • To overcome this problem, electrophoresis is performed with reasonable power settings, to give acceptable separation times and an appropriate cooling system to remove liberated heat
- 14. Types of electrophoresis • Different types of electrophoresis are designed depending upon whether it is carried out in presence or absence of supporting media 1. Based on buffer system 2. Based on support media
- 15. Types of electrophoresis Moving boundary zone Gel Polyacrylamide Non dissociating (Native PAGE) Dissociating (SDS-PAGE) Agarose Paper
- 16. Line diagram of electrophoresis
- 17. Moving boundary electrophoresis • Carried out in u-shape tube with platinum electrode attached to the end of both arms • At the respective ends ,refractometer attached • Sample dissolved in the buffer & loaded in middle of the tube, then apparatus connected to the power supply • Charged molecule move to their respective counter charged electrode & passes through the refractometer • Refractometer measures the change in refractive index of the buffer due to presence of molecule • As desirable molecule is passed, sample is taken out from the apparatus along with buffer Application- Used to study the behaviour of the molecule in an electric field • The resolution of technique is low, due to mixing of the sample as well as overlapping of the sample components
- 18. Zone Electrophoresis • An inert polymeric support media is used in between the electrode to separate and analyze the sample Supporting media- Adsorbent paper Gel of starch Agar Gel Poly acrylamide Gel Media minimizes mixing of the sample Analysis and purification of molecule from the gel much easier than moving boundary electrophoresis Example-Gel Electrophoresis
- 19. Gel Electrophoresis • Vertical gel electrophoresis -- performed in discontinuous way with buffer in upper and lower tank connected by the gel slab • Multiple modification in the running condition to answer multiple analytical problems
- 20. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) • Most widely used method for analysing protein mixture qualitatively • Based on the separation of proteins according to size so particularly useful for monitoring protein purification • Also be used to determine the relative molecular mass of proteins • Instrumental detailing- Two buffer chamber, upper and lower chamber Both chamber fitted with platinum electrodes connected to the external power supply, which supplies a direct current Upper and lower chamber filled with the running buffer, connected by the electrophoresis gel casted in between two glass plates(rectangular & notched) Additional accessories like comb, spacer, gel caster etc. required for casting the polyacrylamide gel
- 21. Fig 4-Schematic diagram of vertical gel electrophoresis Figure 5: Different steps in performace of vertical gel electrophoresis to resolve sample.
- 22. Casting of the gel Combination of two gels the top one is stacking gel(pH 6.8) with lower % of acrylamide and bottom one is Separating or resolving gel(pH 8.8) with higher concentration of acrylamide
- 23. Buffer and Reagents for PAGE • N,N,N’,N’-tetramethylethylenenidiamine(TEMED)-catalyzes acrylamide polymerization • Ammonium persulphate(APS)-initiator for acrylamide polymerization • Tris HCL-component of running & gel casting buffer • Glycine-component of running buffer • Bromophenol blue-tracking dye used for monitoring the progress of the gel • Coomassie brilliant blue R250-used to stain PAGE • Sodium dodecyl sulphate- denature and provide negative charge to the protein • Acrylamide-monomeric unit for gel preparation • Bis-acrylamide-cross linker for polymerization of acrylamide monomer to form gel
- 27. Fig 6: SDS-PAGE setup
- 28. Detailed view of SDS-PAGE
- 29. Running of the gel • Sample prepared in loading dye containing SDS, a Marcaptoethanol in glycerol(facilitates the loading of the sample in the well) • Marcaptoethanol reduces any disulphide bond of tertiary structure & SDS denature the protein • Sample first loaded on stacking gel(1cm) placed above main separating gel(10cm) • Stacking gel concentrate the protein sample into sharp band before it enters the main separating gel • pH of stacking gel-6.8 & very large pore size(4 % polyacrylamide) • pH of seprating gel-8.8 & pore size depends on size of protein being studied • A pure protein give a single band on the SDS-PAGE & two band obtained in case of molecule having two sub units
- 30. Application of vertical electrophoresis 1. Determination of molecular weight- The value of Rf and log molecular weight of the standard protein used to draw the calibration curve to calculate the Molecular weight of the unknown sample. Rf=Migration of protein from the lane/migration of tracking dye a
- 31. 2.Determination of oligomeric status of the protein- • Polyacrylamide gel used to determine oligomeric status of protein • A protein sample electrophoresed under the denaturating as well in the native condition in two separate gel. • Standard protein of known molecular weight also electrophoresed on both gel • A Rf value is calculated as described above and a calibration curve from the native and denaturating gel is used to determine the M.Wt of the protein • Oligomeric status=molecular weight(native)/molecular weight(SDS-PAGE)
- 32. 3.Studying protein folding and unfolding- • In urea PAGE, a polyacrylamide gel prepared with a horizontal gradient of urea(0-8M) • Same protein sample loaded in different lane and allowed to run vertically, perpendicular to urea gradient. In different lane ,sample exposed to different concentration of urea • At a particular urea concentration, the protein is unfolded with an increase in hydrodynamic volume. Unfolded protein migrate slower due to increase in friction force • It gives a unique protein band pattern to provide qualitative or semi-quantitative information about the protein folding intermediate • Urea PAGE also used to analyze the protein complexes as well as covalent hydrogenecity of the protein
- 33. 4.Purification of antigen- • Preparative SDS PAGE used to purify antigen(protein) to generate antibody. Protein of interest produced in large quantity in suitable expression system • Crude lysate resolved in a mini or maxi gel containing a fused lane to load large amount of (2-3ml) of lysate single lane of gel analysed either by staining or with western blotting • Gel portion containing the desired protein cut down and protein is eluted from the gel • Protein is lyophilized and used for immunization of animal such as rabbit Fig8 -purification of antigen
- 34. 5.protein-protein interaction Two approaches are used to study protein-protein interaction In 1st approach, a complex made by incubation of protein A & protein B • Analysis of complex on native PAGE In 2nd approach, first resolution of protein A on the SDS-PAGE Transferred to nitro-cellulosed membrane ,which is blocked with 1% BSA over night at 4 degree Celsius and Incubate protein B with this nitro-cellulose membrane Rinse membrane with buffer followed by probing with anti-B antibody & HRP coupled with secondary antibody(anti IgG- HRP) and Diamino-benzidine (DAB) used to develop blot
- 35. Horizontal Gel Electrophoresis • Performed in a continuous fashion with both electrode & gel cassette submerged within the buffer • Electrophoresis chamber fitted with two Pt electrodes placed on both ends connected with external DC power supply • Chamber filled with running buffer & gel cassette submerged inside the buffer • Additional accessory like gel caster, comb, spacer etc are required for gel casting Fig 10:Different component of horizontal gel electrophoresis apparatus Fig 11: Different steps for casting Agarose gel for horizontal gel electrophoresis apparatus
- 36. Buffer & Reagent for Horizontal Gel Electrophoresis • Agarose - polymeric sugar used for preparation of horizontal gel for DNA analysis • Ethidium bromide - for staining of the Agarose gel to visualize the DNA • Sucrose - used for preparation of loading dye • Tris-HCl - component of running buffer • Bromophenol blue - tracking dye to monitor the progress of electrophoresis
- 37. Casting of the Agarose gel • Agarose powder dissolved in TAE or TBE buffer & heated to melt the agar • Hot agar poured into the gel cassette and allowed to set • A comb inserted in to hot agarose to cast the well for loading sample • In some cases, ethidium bromide added in to the gel so that it stains the DNA while electrophoresis Galactose + 3,6 Anhydrogalactose (agarose)
- 38. Side view of Agarose gel electrophoresis setup
- 39. Running & staining • Whole apparatus set a constant voltage • DNA runs from negative to positive end & EtBr present in the gel stain the DNA • In UV chamber ,DNA shows orange coloured fluorescence
- 40. Application of horizontal gel electrophoresis 1.Determination of size of DNA- determined by comparing the size of the known molecule • Known DNA resolved on 0.8% Agarose along with the unknown sample • The value of relative migration(Rf) of each DNA band is calculated from the Agarose gel • The value of Rf and size of DNA is used to draw the calibration curve to calculate the size of unknown DNA samples
- 41. 2.DNA-Protein interaction • The size and hydrodynamic volume of DNA changes in DNA- Protein complex • A fix amount of DNA incubated with the increasing concentration of protein As complex formed ,hydrodynamic volume of the complex increases ,a shift in band observed Gradual shift in band observed until the DNA binding site is not saturated with the protein molecules
- 42. 3.Electroelution • Seprated protein in polyacrylamide is revived by electro-elution for further usage • Desired portion of the gel block cut down from the SDS-PAGE & placed in the dialysis bag & sealed from the both ends • Selection of dialysis bag based on protein of interest. Dialysis bag placed in horizontal gel apparatus & electrophoresis is performed with a constant voltage • Salt & other molecule contamitant moves out of dialysis bag but protein remained trapped within dialysis bag • Recovered protein further purify using downstream processing Fig 12-electroelution using horizontal gel electrophoresis
- 43. 4.Southern blotting • DNA fragments obtained after restriction endonuclease digestion are resolved using Agarose gel • Alkaline solution used to denature dsDNA in to ssDNA in the gel • DNA transferred from the gel to the nitrocellulose membrane by applying suction pressure or by placing wet paper towel • Incubate membrane with non- specific DNA to block the binding site on the membrane • A single standard probe is also allowed to bind DNA • Membrane is washed & probe is developed by using autoradiography • The DNA fragment complementary to the probe sequence binds and give positive signal
- 44. • Fig 13- Southern blotting using using horizontal gel electrophoresis appratus
- 45. Horizontal Polyacrylamide Gel Electrophoresis • Used for resolving complex biological sample as protein and DNA moved to counter charged electrode • Sample loaded in the middle of the well & get resolved based on their mass/charge ratio • The horizontal native PAGE separates protein mixture with high resolution & protein migration correlate well with mass/charge ratio
- 46. Instrumentation & casting of native PAGE • The gel cassette consist of one big plate & two small plate • A 2mm thick glass plate sticked on the big plate to give in build spacers • Gel cassette assembled with the help of binder clip, a 1cm gap allowed for glass comb • Gel cassette placed in vertical position & acrylamide solution poured in the cassette through 1 cm gap and this form resolving gel • Water equilibrated with butanol over-layered on top of resolving gel • Same procedure allowed to cast resolving gel on other side of glass plate • Gel cassette placed horizontally & stacking gel is poured & comb is placed to cast the wells
- 47. Sample preparation & electrophoresis • Protein samples are mixed with 5x loading dye, 40% sucrose ,10% BPB & 10% MB • BPB, an anionic dye used to monitor mobility of proteins on the anodic side where as MB ,a cationic dye used to track movement on the other side (cathode) of the gel • Gel cassette placed in horizontal direction in chamber ,filled with chilled 1x native tris glycine running buffer • Sample(up to 20μl) loaded in to the well
- 48. Acrylamide, Bis Acrylamide & Polyacrylamide 48
- 49. Fig 14-Design of horizontal gel cassette
- 50. Fig 15: Sepration of protein using Native-PAGE
- 51. Advantages of native PAGE • In conjugation with SDS-PAGE,horizontal PAGE used to seprate & analyze complex biological sample • User friendly & no specialized equipment required • Native preparative gel to purify proteins in bulk for activity assay,antibody development etc
- 52. Conclusion • It involves separation of a components in a sample by the differential rate of migration of ions by attraction or repulsion in an applied DC electric field • Under the influence of an electric field these charged particles migrate either to the cathode or to the anode, depending on the nature of their net charge
- 53. References • Altria ,K.D ,Capillary Electrophoresis Guide Book,Human Press ,1996 • Andrews,Electrophoresis Theory,Techniques & Biomedical Application,Oxford university Press, 1986 • Dunn,M.J,Gel Electrophoresis:Proteins,Bios Scientific ,1993 • Walker,J.M,The Protein Protocols Hand Book,Humana Press,1996 • NPTEL lecture series • Web reference-www.google.com