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Premium member Presentation Transcript ELECTROPHORESIS: ELECTROPHORESIS BY: DR RAVI JAIN Electrophoresis : Electrophoresis Principle -The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biologic molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionizable groups and, therefore, at any given pH, in solution as electrically charged species either as cations(+) or anions(-) Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.THEORY: THEORY If in an electric field of strength ‘E’ a molecule of charge q is present, then the force on the particle causing it to accelerate is F=E q This is balanced by the frictional resistance to give a terminal velocity v. E q=fv Where f is the frictional coefficient Frictional force depends on- Viscosity of the medium Shape and size (r) of the particle. Charge on the moleculePowerPoint Presentation: Electrophoretic mobility it is the ratio of velocity of the ion to the field strength. The distance migrated by the ions will be proportional to both current and time. During electrophoresis the power (w, watts) generated in the supporting medium which is dissipated as heat. Heating of the electrophoretic medium has the following effect. An increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples. Formation of convection currents leading to mixing of separated samples Denaturation of proteins or loss of activity of enzymes. Decrease of buffer viscosity and hence a reduction in the resistance of the medium To avoid this heat effect following measures are to be taken- Reasonable power setting. Appropriate cooling system. .Factors affecting migration of charged particles: Factors affecting migration of charged particles Charge time Voltage Distance between the electrodes Ionic strength of the buffer pH of the buffer At pH 8.6 all serum proteins bear a net negative charge and move towards the anode. If the pH of the buffer is adjusted below 4.8 then all the proteins will move towards the cathode, since they carry positive charge. Size of the molecule- smaller the size faster the movement at a given charge. Among the serum proteins, albumin is the fastest moving component followed by α 1, α 2, ß and ý globulin. Electroendosmosis - due to the presence of charged groups on the surface of the support medium.( Paper –carboxyl group, agarose- sulphate group, glass wall of capillary- silanol group)Electrosmotic flow: Electrosmotic flowInstrumentation: Instrumentation The equipment required for electrophoresis consist basically of two items- a power pack and an electrophoresis unit. An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the support is stained to visualize the separated componentsSchematic of zone electrophoresis apparatus : Schematic of zone electrophoresis apparatusVertical gel apparatus: Vertical gel apparatus Used for separating proteins in polyacrylamide gel.Horizontal apparatus : Horizontal apparatus Used for immunoelectrophoresis, isoelectric focusing and the electrophoresis of DNA and RNA in agarose gel.Support media: Support media Advantages- Porous mechanical supportwhich is wetted in electrophoresis buffer and in which electrophoresis of buffer ions and samples could occur. Cuts down convection current and diffusion providing sharp zones of separated components. Commonly used support media- Filter paper or cellulose acetate strips-for resolving small molecules. Gels- 1) Starch gel 2) agarose gel or polyacrylamide gel.Agarose gel-: Agarose gel- Structure - agarose is a linear polysaccharide made up of basic repeat unit agarobiose , which comprises alternating units of galactose and 3,6- anhydrogalactose Agarose is generally used at a concentration of 1% & 3%. Formation - Pore size- depends on the initial concentration- Lower the concentration, larger will be the pore size. Purity grade- depends on the sulphate content-lower the sulphate content, higher the purity. Uses - electrophoresis of proteins and nucleic acids Agarose gels are used in techniques such as immunoelectrophoresis or flat bed isoelectric focusing , where the proteins are required to move unhindered in the gel matrix according to their native charge. Advantage -low melting temperature agarose- gels can be reliquified by heating to 65 C and thus for example DNA samples separated in a gel can be cut out of the gel, returned to solution and recovered.Polyacrylamide gel- PAGE: Polyacrylamide gel- PAGE Structure - crosslinked polyacrylamide gels are formed from polymerization of acrylamide monomer in presence of small amount of N N’ methylene bis acrylamide( bis acrylamide ) Formation - 1) polymerization of acrylamide is an example of free radical catalysis and is initiated by NNN’N’ tetrametylenediamine(TEMED) & ammonium persulphate . TEMED catalyzes the decomposition of persulphate ion to give free radicle R’ R’ + M = RM’ RM’+M = RMM’ & so on forming long chain & then cross linked by introduction of occasional bis-acrylamide molecule. 2 ) photopolymerization- ammonium persulphate and TEMED are replaced by riboflavin and when gel is poured it is placed in front of a bright light for 2-3 hours . Photodecomposition of riboflavin generates free radical that initiates polymerization.: Acrylamide gels are defined in terms of total percentage of acrylamide present, and the pore size in the gel can be varied by changing the concentration of both acrylamide and bis- acrylamide. Pore size- Low % gel- large pore size for free movement of proteins by electrophoresis without frictional effect in flat bed isoelectric focusing, stacking gel system of SDS-PAGE. 10-20% gel- where smaller pore size introduces a seiving effect that contributes to the separation of protein according to their size.Electrophoresis of proteins-: Electrophoresis of proteins- SDS-PAGE-(sodium dodecyl sulphate PAGE) Most widely used method for analyzing protein mixture. Useful for monitoring protein purification . Separation of proteins according to their size. Determine the relative molecular mass of proteins. Samples to be run on SDS-PAGE are firstly boiled for 5 min. in sample containing β -mercaptoethanol and SDS. Mercaptoethanol reduces disulfide bridges present on proteins. SDS binds strongly and denatures protein- protein opens up in a rod shaped structure. The original native charge on the molecule is therefore completely swamped by negatively charged SDS moleculesPowerPoint Presentation: Sample buffer also contains- 1) An ionizable tracking dye , usually bromophenol blue that allows the electrophoretic run to be monitored. 2) Glycerol or sucrose -gives sample solution density thus allowing the sample to settle easily through electrophoresis buffer to the bottom when injected into the loading well. The samples to be run is not infact loaded directly into the separating gel.Instead they are poured into the stacking gel . The purpose of this stacking gel is to concentrate the protein sample into a sharp band before it enters the main separating gel. This is achieved by utilizing differences in ionic strength and pH between the electrophoresis buffer and the stacking gel and involves the phenomenon known as ISOTACHOPHORESIS .PowerPoint Presentation: Band sharpening effect- electrophoretic mobility Cl > protein-SDS complex > glycinate Glycinate can move at same speed only if they are in a region of higher field strength Field strength is inversely proportional to conductivity, which is proportional to concentration. The result is that the three species of interest adjust their concentration so that (Cl) > (protein-SDS complex) > ( glycinate) There is only a small quantity of protein-SDS complexes, so they concentrate in a very tight band between glycinate and Cl boundaries. Once the glycinate reaches the separating gel it becomes more fully ionized in the higher pH environment and its mobility increases Thus the interface between the glycinate and chloride ion leaves behind the protein-SDS complex, which are left to electrophorese at their own rates.PowerPoint Presentation: Now the protein-SDS complex pass through the separating gel owing to molecular seiving property of gel . Smaller the protein more easily it can pass through the pores of the gel. Being smaller the bromophenol blue dye is totally unretarded and indicates the electrophoresis front When the dye reaches the bottom current is turned off. Gel is removed between the glass plates and shaken in an appropriate stain solution-usually Coomassie Brilliant Blue , for few hours and then washed in destain solution overnight. Stained proteins are visible as blue bands on a clear background. A typical gel would take 1 – 1.5 hours to prepare and set, 3 hours to run at 30mA and have staining time of 2-3 hours with an overnight destainTwo SDS-PAGE gels after a completed run: Two SDS-PAGE gels after a completed runPicture of SDS-PAGE, molecular marker is on left lane.: Picture of SDS-PAGE, molecular marker is on left lane.PowerPoint Presentation: Native (buffer) gels- This is used to detect a particular protein (often an enzyme) on the basis of its biological activity , because the protein is denatured by the SDS-PAGE procedure. In native or buffer gels, polyacrylamide gels are again used (normally a 7.5% gel) but the SDS is absent and the proteins are not denatured prior to loading. Since all the proteins in the sample being analyzed carry their native charge at the pH of gel (normally 8.7), proteins separate according to their different electrophoretic mobilities and the seiving effects of gel. Gradient gels- This is again a polyacrylamide gel system, but instead of running a slab gel of uniform pore size throughout a gradient gel is formed, where the acrylamide concentration varies uniformly from typically, 5% at the top of the gel to 25% acrylamide at the bottom of the gel . Advantage - Much greater range of protein Mr values can be separated. 2) Proteins with very similar Mr values may be resolved, although they cannot otherwise be resolved in fixed percentage gels. Isoelectric focusing gel-: Isoelectric focusing gel- Ideal for separation of amphoteric substances such as proteins because it is based on separation of molecules according to their different isoelectric points pI - IsoElectric Point : pH at which a protein has a net 0 charge (positive and negative charges balance). Depends mostly on the amino acid composition and a little on the tertiary structure . Separation is achieved by applying a potential difference across a gel that contains a pH gradient. The pH gradient is formed by introduction into the gel of compounds known as ampholytes , which are complex mixtures of synthetic polyamino-polycarboxylic acids. Commercially available ampholytes include Bio-lyte and pharmalyte . This method requires the proteins to move freely according to their charge under the electric field, IEF is carried out in low percentage gels to avoid any seiving effect within the gel.PowerPoint Presentation: Following electrophoresis the gel is not stained directly because the ampholytes will stain too, giving a totally blue gel. The gel is first washed with fixing solution ( 10% v/v TCA ).this precipitates proteins in the gel and allows the much smaller ampholytes to be washed out. The gel is stained with CBB and then destained. IEF is a highly sensitive analytical technique and is particularly useful for studying microheterogeneity in a protein. Paritcularly useful for separating isoenzymes . Two-dimensional polyacrylamide gel electrophoresis- : Two-dimensional polyacrylamide gel electrophoresis- This technique combines the technique of IEF , which separates proteins in a mixture according to charge (pI), with the size separation technique of SDS-PAGE .PowerPoint Presentation: A typical 2-D gel . Final gel has a complex mixture of proteins separated by pI along the horizontal axis and by log M along the vertical axisCellulose acetate electrophoresis: Cellulose acetate electrophoresis Used in clinical analysis of serum samples . Advantages over paper electrophoresis- Much more homogenous medium. Uniform pore size Does not adsorb proteins in the way Much less trailing of protein bands. Resolution is better. Simpler to setup and run. Cellulose acetate is first wetted in electrophoresis buffer (pH 8.6 for serum samples and the sample is loaded as a 1 cm wide strip about 1/3 of the way along the strip. The ends of the strip make contact with electrophoresis buffer tanks via a filter paper wick that overlaps the end of the cellulose acetate strip Following electrophoresis , the strip is stained for protein, destained and the bands visualised. A typical serum protein separation shows six major bandsDetection, estimation, and recovery of proteins in gels: Detection, estimation, and recovery of proteins in gels The most commonly used general protein stain for detecting protein on gels is the sulphated trimethylaminedye Coomassie Brilliant Blue R-250 (cbb). Staining is carried out using 0.1% (w/v) CBB in methanol: water: galial acetic acid (45:45:10, by vol.) This acid methanol mixture acts as a denaturant to precipitate or fix the protein in the gel, which prevents the protein from being washed out whilst it is being stained. Staining of most gels is accomplished in about 2h and destaining usually overnight , is achieved by gentle agitation in the same acid methanol solution but in the absence of dye CBB is not used for staining cellulose acetate because it binds strongly to the paper. In this case, proteins are first denatured by brief immersion of the strip in 10% (v/v) TCA, and then immersed in a solution of a dye that does not stain the support material. ( Procion blue, Amido black or Procion S )PowerPoint Presentation: The silver stain is atleast 100 times more sensitive than Coomassie Brilliant Blue, detecting proteins down to 0.1 ng amounts. Quantitative analysis- Densiometry- passing the stained gel track over a beam of light (laser) and measuring the transmitted light. A graphic presentation of protein zones (peaks of absorbance) against migration distance is produced, and peak areas are calculated. 2) An alternative and cheaper way of obtaining such data is to cut out the stained bands of interest, elute the dye by shaking overnight in a known volume of 50% pyridine, and then to measure spectrophotometrically the amount of colour released. 3) Video imaging unitElectrophoresis of nucleic acids: Electrophoresis of nucleic acids Agarose gel electrophoresis of DNA . For majority of DNA samples, electrophoretic separation is carried out in agarose gels. The larger pore size of an agarose gel is required. Since the charge per unit length in any given fragment of DNA is the same, all DNA samples should move towards anode with the same mobility under an applied electric field. However, separation in agarose gels is achieved due to resistance to their movement caused by the gelmatrix. No stacking gel is needed. Most common reagent used for staining is fluorescent dye ethidium bromide. DNA sequencing gels- When DNA sequences are to be determined.PowerPoint Presentation: . Well 2 - DNA digested by restriction enzyme Eco R1 (pink tube) Well 3 - DNA digested by restriction enzyme Hin d111 (green tube) Well 4 - DNA digested by both restriction enzymes (green tube D)PowerPoint Presentation: Pulsed field gel electrophoresis- The agarose gel methods for DNA can fractionate DNA of 60 kb or less. The introduction of pulsed field gel electrophoresis (PEGE) and the further development of variations on the basic technique now means that DNA fragments upto 2000 kb can be separated. This therefore allows the separation of whole chromosome by electrophoresis. Electrophoresis of RNA- Like that of DNA, electrophoresis is usually carried out in agarose gels , and the principle of separation, based on the size is the same.Capillary electrophoresis-: Capillary electrophoresis- Performing electrophoresis in small-diameter capillaries allows the use of very high electric fields because the small capillaries efficiently dissipate the heat that is produced. Increasing the electric fields produces very efficient separations and reduces separation times . Capillary electrophoresis can be used to separate a wide spectrum of biological molecules including amino acids, peptides, proteins, DNA fragments (synth. Oligonucleotides) and nucleic acids. Haemoglobin electrophoresis : Haemoglobin electrophoresis Haemoglobin electrophoresis at alkaline pH- Electrophoresis at alkaline pH( 8.5) using Tris EDTA- borate buffer. Various support media- filter paper, starch gel, or cellulose acetate membranes. Most widely used- cellulose acetate ‘coz- method is simple only small quantity blood is required separation of haemoglobin is rapid quantitation of haemoglobin is possible strips can be stored permanently. Principle - different haemoglobin have different net charge because of variation in their structure. In an alkaline buffer solutions haemoglobins migrate from cathode(-) to anode(+). Different haemoglobins have different rates of migration due to differences in their charge. Haemoglobins having more positive charge than HbA are nearer the cathode while haemoglobins having more negative charge are nearer the anode in relation to HbA. Identification of different haemoglobins is based on their relative positions on cellulose acetate strips.Cellulose acetate electrophoresis at alkaline pH: Cellulose acetate electrophoresis at alkaline pH Lane 1-control (AFSC);prepared by mixing blood from normal infant and persons with sickle cell trait and Hb C trait. Lane2- AA (normal person); lane3- SS (sickle cell anaemia); lane4-AS (sickle cell trait.Citrate Agar Electrophoresis at acidic pH: Citrate Agar Electrophoresis at acidic pH Useful method for further characterization of haemoglobin variants after electrophoresis at alkaline Ph Lane 1; control ( A, F,S,C); Lane 2; AA ( Normal) Lane 3 : SS ( sickle cell anemia) Lane 4; AS ( sickle cell trait)THANKS: THANKS BY: DR RAVI JAIN Assistant Professor Department of Pathology M.G.M MEDICAL COLLEGE, INDORE You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.