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Electrophoresis.pptx
- 1. Electrophoresis Dr Keyur Madhu MDBiochemistry Assistant Professor Shantabaa Medical College, Amreli
- 2. • It isa type of separative procedure widely used in clinical & research laboratories • Used to separate complex biomolecules like Proteins, Amino acids , Peptides, Carbohydrates, DNA fragments, RNA etc.
- 3. • Electro –Refers to the energy of electricity • Phoresis – From the Greek verb “Phoros” means to “carry across” • Migration of charged particles or solutes of any size in a liquid medium under the influence of an electric field
- 4. • Biological moleculessuch as Amino acids, proteins, peptides, nucleotides & nucleic acids possess ionisable groups • Hence depending on the pH, they exist as charged molecules in the solution either as cation(+ve ly charge) or anions(-ve ly charged) • Under the influence of an electrical field charged particles will migrate either to cathode or anode depending on the net charge they carry
- 5. •So it isobvious that molecules having similar charges move in the same direction • But because of their difference in the molecular mass the extent to which these they move differs • Hence the difference in the Charge : mass ratio (C/M) forms the basis of differential migration of particles in an applied electric field • And this forms the general principle of electrophoresis
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- 7. • When charged moleculesare placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. 7
- 8. Mice run fasterthrough the forest than elephant 8 1 2
- 10. (a). A typicaldiagram of vertical electrophoresis. (b). Sample after electrophoresis
- 12. 1. The Electricfield • Voltage & Current – rate of migration is directly proportional to the voltage & current applied • Resistance – migration rate is inversely proportional to resistance 2. Sample • Charge – Migration rate increases with increase in the net charge • Size – As molecule size increases, migration rate decreases 3. Buffer • Composition – It should be such that buffer does not bind with the compounds to be separated as it may alter the rate of migration
- 13. • Concentration –As the ionic strength of buffer increases, the proportion of current carried by the buffer increases and the current carried by the sample decreases thus slowing down the rate of migration • pH – Determines the extent of ionization and thus the degree of migration will be pH dependent 4. Supporting medium • Viscosity – Increase Viscosity slows migration • Pore size - Increase pore size increase in the rate of migration 5. Temperature • Increase in temperature increases the heat & this increases the rate of migration But this will denature the sample
- 14. 1. Depending onthe nature of supporting medium A. Agarose Gel Electrophoresis B. Polyacrylamide gel electrophoresis C. Cellulose acetate electrophoresis D. Capillary electrophoresis 2. Depending on the mode of technique A. Disc electrophoresis B. Isoelectric focusing electrophoresis C. Two-dimensional Polyacrylamide gel electrophoresis D. Pulse field electrophoresis E. Immunoelectrophoresis
- 15. 1. Depending onthe nature of supporting medium AGAROSE GEL ELECTROPHORESIS: • It is a widely used method that separates molecules based on charge, size & shape • Used in Biochemistry & Molecular biology to separate a mixed population of DNA & RNA fragments by length or to separate proteins by charge • Gels can be run using horizontal or vertical system
- 16. Reagents required Agarosepowder Buffer Sample Tracking dye - Bromophenol blue Fixative Staining & destaining solution Distilled water
- 17. Serum proteins: Amido Black – 640 nm Coomasie Brilliant Blue G250 - 595nm Coomasie Brilliant Blue R250 - 560nm Ponceau S – 520 nm Silver stain Isoenzymes : Nitrotetrazolium Blue-570 nm
- 18. Lipoprotein zones : SudanRed 7B - 540 nm Oil Red O - 520 nm Coomasie brilliant blue R250 - 560nm DNA Fragments : Ethidium bromide Nucleic acids : Silver stain Ethidium bromide CSF Proteins : Silver nitrate
- 19. Albumin => α₁-globulins => α₂-globulins => β₁-globulins => β₂-globulins => γ-globulins=> Typical result of an electrophoretic separation of human proteins Including densitometric evaluation
- 20. POLYACRYLAMIDE GEL ELECTROPHORESIS : •Most widely used method in Biochemistry, Forensics, Genetics and Molecular biology to separate proteins according to their size • Bcoz the method is based on separation of proteins according to their size, it can also be used to determine molecular mass of proteins • Two types of PAGE exist : • Native PAGE • Denaturing PAGE/SDS PAGE
- 21. NATIVE PAGE : •Proteins are not denatured and therefore separated based on their charge to mass ratio • Since proteins being analyzed carry their native charge at the pH of the gel, proteins separate according to their electrophoretic mobilities and the sieving effect of the gel • One downside is that complexes may not separate clearly or predictably, since they cannot move through the PAG as quickly as individual denatured proteins
- 22. SDS (Sodium DodecylSulfate)-PAGE ELECTROPHORESIS : • Most desirable method for qualitative analysis of the protein mixtures • Proteins are denatured by SDS, this provides net uniform charge to the molecules • Proteins are separated according to their size. • USES : • To assess purity of proteins • To determine MW of Proteins
- 24. CELLULOSE ACETATE ELECTROPHORESIS •Bcoz of higher resolution, it is used to separate so enzymes, lipoproteins Hemoglobins & for Immunoelectrophoresis • It is a homogenous medium with a uniform pore size and does not adsorb proteins => less trailing of protein bands & resolution is better
- 25. INTERPRETATION OF HbELECTROPHORESIS • Normally Hbs present in adults are Hb A, Hb A₂ and Hb F • Hb A comprises 98%, Hb A₂ - 2.5% Hb F - < 1% • Hb A migrates the fastest, followed by Hb F and Hb A₂ moves only slightly from the point of origin near the cathode
- 26. ABNORMAL HEMOGLOBINS-showthe following migration pattern : • Hb C migrates with Hb A₂ near the cathode • Hb S lies between Hb A₂ and Hb F • Hb H and Bart’s hemoglobin are unstable and very fast moving placing them past Hb A and near the anode with Hb H being the fastest of the two • Diagram shows relative migration of hemoglobin variants :
- 28. CAPILLARY ELECTROPHORESIS : •Carried out in a buffer filled small bore fused silica capillary tube of 20 -200 cm in length APPLICATIONS : • Used to separate wide spectrum of biological molecules including amino acids, Peptides, proteins, DNA fragments, Oligonucleotides, Nucleic acids, drugs,vitamins & carbohydrates.
- 29. Isoelectric focusing • Thistechnique takes the advantage of the fact that each protein has a different isoelectric pH(pI) i.e the pH at which it is electrically neutral and does not move the electrical field. • The gels are impregnated with the ampholite that makes continuous pH gradient in the polyacrylamide gels. • The proteins migrates in the electrical field according to their charge : weight ratio and stop migrating as soon as they reach their respective pI.
- 30. • The focussingof the proteins to their pI makes the bands very sharp. • This is a very powerful method and capable of detecting proteins that differ in their pI values by as little as 0.01 units.
- 31. Two-dimensional (2-D) gel electrophoresis •Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two- dimensional Electrophoresis • Two-dimensional electrophoresis separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weights.
- 32. Immune-electrophoresis • In thistype of electrophoresis, antigen & antibody are allowed to interact in the agarose gel. • First the electrophoresis is carried out by applying the patients serum into the wells. • To visualize them, a specific antibody is placed in the gel and incubated. • The precipitation arcs are formed where antigen &antibodies are in1:1 ratio.
- 33. CLINICALAPPLICATIONS OF ELECTROPHORESIS •Themost common uses of electrophoretic techniques in the laboratory are : 1. Specific protein electrophoresis: • Quantitative analysis of specific serum Protein classes such as gamma globulins and albumin • Identification & quantitation of hemoglobin and its subclasses • Identification of monoclonal proteins such as Bence Jones Gamma globulin in either serum or urine • Separation & quantitation of major lipoprotein classes
- 34. 2. ISOENZYME ANALYSIS •Separation & quantitation of enzymes such as creatine kinase, lactate dehydrogenase & alkaline phosphatase into their respective subtypes 3. Used to determine qualitatively the elevation or defiency of specific classes of Immunoglobulins 4. Useful in diagnosing various diseases like Multiple Myeloma, Nephrotic syndrome, sickle cell disease, cirrhosis of liver etc.
- 36. Nephrotic syndrome : •There is increase permeability of glomeruli to proteins so that all proteins except very Big molecules are lost through urine So α₂ fraction containing macroglobulin will be prominent.
- 37. Liver cirrhosis • Albuminsynthesis by the liver is decreased with a compensatory excess synthesis of globulins by reticuloendothelial system. So albumin band will be thin with a wide β-fraction. • Sometimes beta and gamma fractions are fused.
- 38. Acute Inflammation: • Alpha-1and Alpha-2 bands are increased during inflammation due to increased Hepatic synthesis of acute phase reactant proteins
- 39. CHRONIC INFLAMMATION : •Immunoglobulin synthesis by the antigen activated T lymphocytes transformed to Plasma cells is demonstrated by the Polyclonal gamma band.
- 40. MONOCLONAL GAMMOPATHY • Anunusual sharp band in the gamma region suggests the presence of homogenous Immunoglobulin & thus malignant proliferation of plasma cells from a single clone of cells in contrast to the broad gamma band seen in chronic inflammation
- 41. Alpha-1 Antitrypsin deficiency •As alpha -1 antitrypsin is the major component Alpha-1 band there is deficiency of alpha-1 band.