Electrophoretic techniques for life science researchers

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Electrophoresis is a separation  technique that is based on the movement of charged particles in an electric field.
Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field

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Electrophoretic techniques for life science researchers

  1. 1. Electrophoretic Techniques forLife Science ResearchersPresented byDr. B. Victor., Ph.D.,email : bonfiliusvictor@gmail.comblog : bonvictor.blogspot.com
  2. 2. Presentation out line• Origin definition and historical notes• Electromigration, electromotive force and electrophoretic theory.• Types of electrophoretic methods.• Buffers and support media.• Frontal, zonal, starch and agarose gel electrophoresis.• Modifications of gel systems.• SDS-PAGE and DISC- PAGE systems.• Practical applications.
  3. 3. What is electrophoresis? Electrophoresis • Electrophoresis is a separation technique that is based on the movement of charged particles in an electric field.
  4. 4. Meaning of electrophoresis electrophoresis • The term „electrophoresis‟ was coined from the Greek word ‘phoresis‟, which means „being carried‟. • Electrophorosis literally means „to carry with electricity‟.
  5. 5. Historical notes In 1879, Hermann von In 1930, Swedish In 1807,Russian chemist Arne Tiselius Helmholtz generalized physicist Alexander described the first the experimental Reuss identified the electrophoretic system. observations into an migration of colloidal equation of He received the Nobelparticles in an electrical electrophoretic prize in chemistry in field. principles. 1948 for his work.
  6. 6. Definition of electrophoresis Micro -bioanalysis • Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field.
  7. 7. Biomolecules with ionizable groups Amino acids Peptides DNA proteins RNA nucleotides
  8. 8. Electro-migration At any given pH , the electricallycharged molecules may exist in solution either as cations (+) or anions (-). Negatively Positively charged chargedmolecules move molecules moveto the anode(+). to the cathode(-).
  9. 9. Transport Processes in electrophoresis• Electro migration• Negatively charged molecules (anions) move towards the anode (+).• Positively charged molecules (cations) move towards the cathode (-).• Highly charged molecules move faster towards the electrode of opposite charge than those with lesser Electro osmosis• movement of entire fluid near wall of capillary in one direction!• anode (+ve) -> cathode (-ve)
  10. 10. Electromotive force(EMF)• Electrophoresis is based on electromotive force(EMF) that is used to push or pull the molecules through the gel matrix.• By placing the mixture of molecules in wells in the gel and applying an electric current, the molecules will migrate through the matrix.• The separated molecules take directions based on the total electric charges.
  11. 11. Principle of velocity of migration ofseparated moleculesVelocity of migration of the molecules, v=E.q / fWhereE=electric field in volts/cmQ=the total electric charge on the moleculeF=the frictional coefficient which is a friction of the mass and shape of the molecule.
  12. 12. Electrophoretic Theory• Two laws are relevant to the use of power supplies for electrophoresis of macromolecules:• Ohm’s Law and• Second Law of electrophoresis.
  13. 13. Ohm’s Law• Current (I)=Voltage (V)/Resistance (R)• Ohm‟s Law states that current is directly proportional to the voltage and is inversely proportional to the resistance.• Resistance of the system is determined by the buffers used, the type and configurations of the gels being run, and the total volume of all the gels being run.
  14. 14. Second Law• Watts (W)=Current (I) x Voltage (V)• The Second Law states that power or watts (a measure of the heat produced) is equal to the product of the current and voltage.• Since V=I x R, this can also be written as Watts=I2 x R.
  15. 15. Factors influencing rate of migration of ionsNet charge of the moleculeSize and shape of the moleculeBuffer pHStrength of electrical fieldProperties of support mediaTemperature of the operating system
  16. 16. Electrophoresis apparatus Power pack BuffersElectrophoresis Electrophoresis Equipment media Electrophoresis support media unit Electrophoretic chamber
  17. 17. Types of electrophoresis Paper electrophoresis Disc electrophoresis Moving boundary/frontal electrophoresis Column electrophoresis ZonalElectrophoresis electrophoresis Capillary electrophoresis Immuno-electrophoresis Open block electrophoresis Isoelectric focusing
  18. 18. Gel electrophoresis Gel electrophoresis Tube gel Slab gelelectrophoresis electrophoresis vertical slab gel Horizontal slab gel electrophoresis electrophoresis
  19. 19. Buffers Barbitone buffer – • serum protein separation , (around 8.0 pH) • poor resolution, weak buffer. Phosphate buffer- • Enzyme separation, ( around 7.0 pH) • low buffering capacity.- high conductivityTris – borate – EDTA • Nucleic acid Separation buffer (TBE) -(pH • Good resolution , high buffering capacity , low around 8.0) conductivity.Tris – acetate – EDTA • Nucleic acid separation buffer (TAE)- (pH • high resolution , high buffering capacity , around 8.0) low conductivity. • protein separation • highTris – glycine buffer - • Protein separation(pH more than 8.0)- • high buffering capacity , low conductivity
  20. 20. Support media• Paper – poor conductor of electricity absorbate proteins, non - transparent poor resolution.• Agar- flow of solvent electro endosmosis, vary thickness , transparent poor resolution• Cellulose acetate strip- tailing of bands poor resolution non-absorbing.• Starch- form opaque gels non-absorbing high resolution• Agarose -highly transparent porous – high resolution east preparation• Acrylamide – stable , non –reactive highly transparent.
  21. 21. Moving boundary/frontal electrophoresis• Protein solution is injected Tiselius in 1937 introduced into a quartz U-tube. this technique.• The arms of the U- tube are filled with buffers.• When electricity is passed, different species of protein molecules separate in to bands of proteins.• Discrete zones does not occur.
  22. 22. Zonal electrophoresis• Sample is applied as a narrow band. Consden,Gordon and Martin in• Separation occurs discrete bands. 1946 introduced this technique• Numerous support media – paper, cellulose acetate, agar gel starch gel and acrylamide gel can be used
  23. 23. Paper electrophoresis• A small volume of the This technique was introduced by Durrum (1950), Flynn and sample is placed evenly Mayo (1951) along a line drawn across a strip of Whatmann paper previously soaked in buffer.• The ends of the paper are soaked in buffer solutions.• Passage of electricity cause separation.
  24. 24. • Starch matrix is suitable for isoenzymes . Starch gel• Partially hydrolysed potato starch electrophoresis is used.• The gels are slightly more opaque than acrylamide or agarose.• Non-denatured proteins can be separated according to charge and size.• They are visualised using Napthal Black or Amido Black staining.
  25. 25. Cellulose acetate electrophoresis• Strips of cellulose acetate are Kohn (1957-1961) introduced this technique used.• Better resolving power• No absorption of proteins• No trailing• Excellent separation of plasma proteins.• transparent
  26. 26. Gel electrophoresis that involves the use of a gelatinous material such as agarose, acrylamide, starch or cellulose acetate as the matrix.• The gel acts as a support medium for the sample. Gel electrophoresis• Gels are used to separate samples containing proteins or DNA. Electrophoresis through agarose or polyacrylamide gels is a standard Starch Gel -- swollen potato starch granules. method used to separate, identify and Agarose Gel is a natural linear polymer extracted purify nucleic acids. from seaweed that forms a gel matrix by hydrogen- bonding when heated in a buffer and allowed to cool. Polyacrylamide Gels -Polyacrylamide gel is made chemically by acrylamide (the monomer) and bisacrylamide (the cross-linker) catalyzed by initiator (amonnium persulfate or riboflavin) and accelerator (TEMED). Acrylamide can be polymerized into any desired shape :• Tube Gels -- polymerize in glass tubing ==> cylindrical shape• Slab Gels -- polymerize between glass plate
  27. 27. Uses of gel electrophoresis• Human DNA can be analyzed to provide evidence in criminal cases, to diagnose genetic diseases, and to solve paternity cases.• Samples can be obtained from any DNA-containing tissue or body fluid, including cheek cells, blood, skin, hair, and semen.• A person‟s “DNA fingerprint” or “DNA profile” is constructed by using gel electrophoresis to separate the DNA fragments from several of its highly variable regions.• Conservation biologists use DNA profiling to determine genetic similarity and kinship among populations or individuals.
  28. 28. • Agarose gel electrophoresis is a powerful separation method Agarose gel electrophoresis frequently used to analyze DNA fragments generated by restriction enzymes.• The separation medium is a gel made from agarose, which is a polysaccharide derivative of agar.• The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon charge, size and shape.• These characteristics,together with buffer conditions, gel concentrations and voltage, affect the mobility of molecules in gels.
  29. 29. Differences between agarose andpolyacrylamide gels Agarose gel Polyacrylamide gel A polysaccharide extracted A cross-linked polymer of from sea weed. acrylamide. Gel casted horizontally Gel casted vertically Non-toxic Potent neuro-toxic Separate large molecules Separate small molecules Commonly used for DNA Used for DNA or protein separations separations. Staining can be done before Staining can be done after or pouring the gel pouring the gel.
  30. 30. Modifications of gel systems Homogeneous • The porosity/percentage of the gel through out the gel system remains same. gel system • Only small molecules can be separated. • It is employed in DISC-PAGE. Heterogeneous • Two different buffers can be used. gel system • All types of molecules can be separated. Gradient • Linear or exponential gradients can be made. • The formation of gradients can be examined by Gel system using dyes.
  31. 31. Gel electrophoresis SDS-PAGE Gelelectrophoresis DISC-PAGE
  32. 32. Sodium dodecyl sulfate-PolyacrylamideGel Electrophoresis – SDS- PAGE -1• SDS-PAGE is a most widely used technique for analysis and characterization of proteins and nucleic acids.• Sample preparation – The protein sample is heated at 1000C in a dilute solution sodium dodecyl sulfate .This breaks down all native quaternary, tertiary, and secondary structures.• Then b-mercapto ethanol is added to cleave the disulfide bonds.
  33. 33. Sodium dodecyl sulfate-PolyacrylamideGel Electrophoresis – SDS- PAGE - 2• Gel preparation – the polymerization is initiated by ammonium per sulfate or riboflavin. N- tetramethyl ethylene diamine (TEMED) catalyses the formation of free radicals from persulfate which in turn initiate polymerization.• Gels ranging from 3 to 30% acrylamide concentration can be made and can be used for the separation of molecules up to 1x106 datons.
  34. 34. Apparatus setup for slab gelelectrophoresis Sample wells Cathode Reservoir Buffer Gel Anode Reservoir Buffer
  35. 35. Sodium dodecyl sulfate-PolyacrylamideGel Electrophoresis – SDS- PAGE - 3• Sample application –about 2 µg of the sample is loaded in each well. Over loading of samples decrease the resolution of bands.• Marker dyes – to follow the sample tracking a marker dye e.g. bromophenol blue gives color. After run the gel was stained with the dye coomasie blue and photographed.
  36. 36. Advantages of polyacrylamide gels Stable over a wide Hydrophilic and Transparent to range of pH,Chemically inert electrically light temperature, and neutral ionic strength. Available in wide Never bind to Superior range of pore proteins. resolution sizes.
  37. 37. Applications of PAGE1. PAGE is used to estimate molecular weight of proteins and nucleic acids.2. PAGE is used to determine the subunit structure of proteins.3. PAGE is used to purify isolated proteins.4. PAGE is used to investigate various liver and kidney diseases by analyzing human serum proteins.5. PAGE is used to monitor the changes in protein content in body fluids.
  38. 38. Continuous - discontinuous gel systems Continuous system--gel and tank buffers are the same, single phase gel; examples are PAGE, agarose, and starch gels. Discontinuous system-- gel and tank buffers are different, two phase gel (stacking gel); example is PAGE.
  39. 39. Discontinuous polyacrylamide gelelectrophoresis –DISC-PAGE - 1• Two gel systems – a stacking gel and a running gel• Several buffer systems- Cathode - Tris – glycine 8.6 pH Wells – Tris – Cl 6.5 pH Stacking gel – Tris – Cl 6.5 pH Separating gel – Tris – Cl 8.7 pH Anode - tris – glycine 8.• Generation of voltage discontinuity
  40. 40. Apparatus setup for DISC - PAGE Cathode Tris – glycine pH 8.3 Buffer Tank Sample in Tris -Hcl , pH 6.7 Stocking gel-Tris –Hcl , pH 6.7 Running gel -Tris – Hcl , pH 8.9 Anode Tris – glycine pH 8.3 Buffer tank
  41. 41. Discontinuous polyacrylamide gelelectrophoresis –DISC-PAGE - 2 Sample application and run – (-) After injecting the sample, the power is turned on and voltage is Large molecules adjusted to 60 mA. Small molecules All -vely charged species start migrating towards anode(+). When marker dye reaches the top of the gel, the power is stopped . The separated zones in gel slab are denatured by acid treatment Glycinate ions (50% TCA) or (7-!) % acetic acid). Bromophenol blue For nucleic acids, ethidium bromide is used for staining. Cl ions For proteins, coomasie blue is (+) used for staining.
  42. 42. Applications of electrophoresis A versatile analytical tool A valuable diagnostic A major tool in the in forensics, moleculartool in clinical pathology- analysis of human DNA biology, genetics,analysis proteins in body in Human Genome microbiology and fluids, Project. biochemistry. A separation tool in A common laboratory serum proteins, tool in the analysis of isoenzymes, proteins, DNAs and immunoglobulin, RNAs. abnormal haemoglobins and serum lipoproteins.
  43. 43.  Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier‟ s College, Palayamkottai, India-627001. He was the dean of sciences, IQAC coordinator and assistant controller of examinations. He has more than 32 years of teaching and research experience He has taught a diversity of college courses and guided 12 Ph.D scholars. Send your comments to : bonfiliusvictor@gmail.com

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