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Electrophoresis

The movement of ions or molecules towards one of the electrodes under the influence of an electric field in a solutions called electrophoresis. Principle Any Substance suspended in water dissociates into charged particles. When these charged particles are subjected to an electric field, all positively charged ions would move towards the cathode or negative electrode and negatively charged ions towards the anode or positive electrode. This is the basic principle involved in electrophoresis

Factors Affecting the Migration of Substances Nature of charged particles The electrophoretic mobility of substances depends on their size (molecular weight and shape therefore, larger particles e!hibit lesser electrophoretic mobility when compared to smaller particles. Smooth"surfaced molecules migrate faster than rough"surfaced substances. Buffers #uring electrophoretic mobility, a buffer solution provides the environment of charged particles, the re$uired p%, electrolytic concentration, ionic strength and viscosity. The choice of the buffer solution depends on the nature of the sample to be separated. The commonly used buffers include titrate, phosphate, &#TA, acetate, Tris, barbitone etc. Ionic Strength Substances with less ionic strength e!hibit a faster separation whereas those with increased ionic strength show a slower separation. pH

The degree of ionization of substances depends on the p% so that their rate of separation is also affected by p%. An increase in p% causes ionization of organic acids and the ionization of organic bases increases in decreased p%.

Electric field The current applied to the electrodes in a solution is carried mainly by the ions of the buffer solution even though some $uantity is carried by the ions of the sample. The rate of movement of ions under potential gradient is called 'mobility'. The increased potential gradient increases the rate of movement of the particles. (n the other hand, resistance plays an important role in the separation of particles. )or instance, the electric current increases when the resistance is decreased and so the separation is faster. *oreover, the buffer ions carrying more charge than the ions of the sample would result in slower separation. Therefore, a constant current is to be maintained by using power pac+ during electrophoresis. Electrodes ,n electrophoretic studies, platinum, carbon or Ag-Ag.l electrodes are used. (f these, platinum electrodes are mostly preferred. Though the use of carbon electrode is ine!pensive, they are easily polarized and re$uire fre$uent replacement. The silver electrodes are to be coated periodically. At each electrophoretic run, the polarity of the electrodes is to be reversed in order to prolong the life of electrodes and buffer solution. Supporting Media in Electrophoresis /arious types of supporting media are used in electrophoretic separation of substances and are as follows. Paper 0aper containing nearly 123 of cellulose with very low adsorption capacity can be used as a stabilizing medium in electrophoresis.

Gel

4els are porous in nature and so the size of the pores in relation to size of the molecule determines the mobility of substances. Starch Though the resolving power of starch gel is very high its pore size cannot be controlled. Agar Agar is soluble in a$ueous buffer solutions and it forms a gel having a large pore size but without molecular sieving. %ence it is used to separate proteins and nucleic acids. Polyacrylamide 0olyacrylamide gel is prepared from components namely 5,56"methylene bisacrylamide(bis ,ammonium persulphate and tetrame7thylene diamine (T&*&# ,,t has low adsorption capacity but has no power of electrosmosis. 8sing differential concentration of the reagents can control the pore size of this gel. Agarose acrylamide ,t is a mi!ed form of gel obtained by mi!ing acrylamide with agarose. %ere acrylamide provides sieve action while the agarose gives physical support to the gel. !ther gels ,n addition to the above types of gels, substances li+e pectin, sephade!, gypsum, polyvinyl chloride, polyvinyl acetate, etc. are also used in electrophoresis, although only rarely. "#PES !F E$E%"&!PH!&ESIS B!'N(A&# E$E%"&!PH!&ESIS This techni$ue is the basis for all modern electrophoretic devices. The apparatus consists of a '8' shaped cell provided with electrodes at the top position of the limbs as shown in the )igure 2.97.

The sample to be separated is dissolved in an appropriate buffer solution and the mi!ture is filled in the cell below the layer of buffer solution. The entire cell is +ept in a temperature bath to have a constant temperature. The p% of the sample is adjusted in such a way that all the molecules of the sample possess a net negative charge. 8nder an electric field between the electrodes, all the molecules move towards the anode from the sample" buffer mi!ture to the region of buffer solution forming a boundary or front. As a result, the refractive inde! of the substance varies along the cell with a steep change at the boundary region. The change in refractive in"dices. .an be measured by optical devices which will e!hibit the direction and the rate of migration of molecules in the sample. %owever all the fractions of the sample are not completely separated by this techni$ue.

)!NE E$E%"&!PH!&ESIS ,n this techni$ue, the charged particles move as zones through a solid medium which may be a paper or powder"coated glass or silica gel. ,n other words, the separated fractions occur in the form of well"defined and permanent zones. Therefore, preservation of zones, interpretation of results and $uantification of separated substances are easy. :one electrophoresis includes two important types namely paper electrophoresis and gel or disc electrophoresis PAPE& E$E%"&!PH!&ESIS Two components containing buffer solution are interconnected by a supporting glass plate. The different buffer solutions used in paper electrophoresis. The level of buffer in the compartments is e$ualized. A suitable filter paper is selected and is made into strips having a width of ;"2 cm. At the center of strip, the origin line is mar+ed with pencil in the form of a spot or a strea+. The paper is now soa+ed in buffer solution and pressed between a blotting paper. The sample is applied on the spot with the help of capillary tube or a micropipette without damaging the paper.

Then the paper is placed on the supporting plate with each end dipped in the buffer solution of the opposite compartment. The electrodes of the compartments are connected to a power supply. The current is applied and the electrophoresis is run for 72 minutes at very low voltage (2"<= /-cm . When the separation is over, the paper is removed, dried and stained with locating reagents. Table 2.> shows various locating reagents used in paper electrophoresis. After elution of fractions using a colorimeter, photoelectric scanning or a densitometer can ma+e the $uantification. The paper electrophoresis used for the separation of substances at low voltage is called low voltage electrophoresis. This method is valuable in the separation of high molecular weight substances li+e proteins, carbohydrates and nucleic acids but it has only limited value in the separation of amino acids. When a high voltage current (9== /-cm for <="<== minutes is used in paper electrophoresis, it is called high voltage electrophoresis which is used for the separation of low molecular weight compounds and smaller ions li+e amino acids and inorganic substances. This techni$ue re$uires a current of high voltage and longer duration (<=== /-cm and <>-97 hours . When the Sample is allowed to flow continuously on the paper by some devices for the separation, then it is called continuous flow electrophoresis. The method is useful in the separation of amino acids and protein %E$$'$!SE A%E"A"E E$E%"&!PH!&FSIS %ere cellulose acetate strips are used instead of paper to separate molecules with better resolution. While using cellulose acetate, the problem of adsorption is eliminated. .ellulose acetate is chemically pure, translucent and. not very hydrophilic. The buffers for this techni$ue are the same as those used in paper electrophoresis. ?ut the solvents such as glacial acetic acid, cotton seed oil, li$uid paraffin, etc. which ma+e cellulose acetate transparent, are more preferred. .ellulose acetate electrophoresis is used to separate glycoproteins, lipoproteins and hemoglobin. ,n addition, immunoelectrophoresis uses cellulose acetate strips. GE$ E$E%"&!PH!&ESIS ,n gel electrophoresis, gels are used as the medium instead of paper. When the gel is pac+ed as a column, it is called column zone electrophoresis. ,f the gel is coated on glass plates, it is called open bloc+ electrophoresis. #isc electrophoresis is a"

column zone electrophoresis because gel is pac+ed as a vertical cylindrical column in tubes. P!$#A%&#$AMI(E GE$ E$E%"&!PH!&ESIS *PAGE+ The apparatus consist of a buffer reservoir system and a power pac+. The buffer reservoir system has an upper cathode compartment and lower anode compartment containing buffer solution. 4lass tubes having <.7 cm length and =.2 cm diameter connect to compartment. The compartment are provided with platinum electrodes. (ne end of each tube is tightly closed with a rubber cor+ and is filled with <.< ml of small pore solution (separation gel . Above this solution, =.< ml of double" distilled water is added and allowed to polymerize for ;= minutes. After the removal of water layer, =.9 ml of large pore solution (spacer gel is overlaid followed by =.< ml of distilled water for polymerization (<2 minutes . Then the water layer is removed and =.=2 ml of the sample is added followed by =.==< ml of mar+er dye. The remaining space of the rube is filled with Tris buffer. After removing the rubber cor+s, the tubes are attached to the compartments in such a way that one end of a tube is attached to the anode compartment and the other end to the cathode compartments are filled with Tris buffer (9== ml and current is applied. When the mar+er dye migrates to the re$uired position in the tube, the electrophoretic run is stopped and the tubes are removed from the compartments.

The gels are immediately removed from the tubes with the help of a syringe needle and each gel is stored @=3 acetic acid contained in a test tube. The gels are stained with appropriate dyes and the sample is $uantified spectrophotometrically or densitometrically. S(S P!$#A%&#$AMI(E E$E%"&!PH!&ESIS *S(S PAGE+ This method is used for the study of the subunits of oligomeric proteins. The oligomeric proteins are formed by more than one polypeptide chain and are stabilized by hydrogen and disulphide bonds and also by hydrophobic interactions. Therefore these proteins move as a single unit during gel electrophoresis. ,n order to separate the subunits of these proteins, the solubilizing agents called solubilisers denature their structure. 8rea, sodium dodecyl sulphate and b mercaptoethanol are mostly used as solubilisers. ,n concentrated urea, the hydrogen bonds readily dissociate. Sodium dodecyl sulphate (S#S , an anionic detergent, disrupts hydrophobic interactions and provides negative charge to the denatured polypeptides. The disulphide bonds are bro+en by mercaptoethanol. ,n S#S"0A4&, S#S incorporated to the gel is used to separate individual polypeptide chains from oligometric proteins. The gels which have the property of molecular sieving e!hibit a linear relationship between the electrophoretic mobility of protein, incorporated to S#S and the molecular weight of proteins. Therefore molecular weight of proteins can also be determined by this method. S$AB GE$ E$E%"&!PH!&ESIS ,n this type, the polyacrylamide gel is allowed to polymerize into a thin slab between two glass plates. At one end of the gel, placing a comb into the gel prior to its polymerization ma+es wells for spotting samples. After polymerization of the gel, the comb is removed and a number of sample wells are got in the form of casts, number of sample can be analyzed and compared at a time. ",! (IMENSI!NA$ GE$ Electrophoresis ,t is a powerful tool and is designed by combining the resolving power of isoelectric focusing with S#S"0A4&. As the molecular weight and isoelectric point of a macromolecule are not related with each other, this techni$ue ma+es use of these two properties to separate the molecules with great resolution power. ?y

this method, a mi!ture of large number of proteins can be resolved into individual fractions. ,n this techni$ue, a protein mi!ture is subjected to isoelectric focussing on gel in a capillary tube. ,n contrast to the conventional electrophoresis in which the p% between anode and cathode is constant, in isoelectric focussing, a p% gradient is maintained with gradual increase from anode to cathode. When the protein sample is introduced into the system at p% below its isoelectric point, it has a net positive charge and will migrate towards the cathode (proteins possess a net positive charge in an acid medium . Therefore, the protein molecule moves to a medium of higher p% due to the presence of p% gradient. %igh p% of the medium influences the ionization and charge of the molecule, so the number of positively charged particles decreases. Thus at a particular p% (isoelectric point , the net charge of the protein becomes zero and will not move further. At this point, the gel is removed from the capillary tube, +ept on the sample wells of a slab gel and subjected to S#S"0A4&. 5ow, the proteins are separated according to their molecular weight. 8ses 4el electrophoresis is widely used to separate and isolate a large number of macromolecules. ,n molecular biology, this is a versatile techni$ue used for determining se$uences of #5A molecules, in verifying nature of nucleic acids and in restriction mapping of #5A. IMM'N!E$E%"&!PH!&ESIS ,t is combined techni$ue of electrophoresis and specific ,mmune reaction. A glass slide is coated with agar or agarose gel and rectangular and round holes are made in the gel. The round wells are filled with two different antigens (<"<== mg and the gel plate is subjected to electrophoresis at low voltage for <"9 hours. Then the plate is removed, from the electrophoretic chamber and the rectangular well is filled with appropriate antiserum. The plate is incubated overnight at room temperature in a moist chamber, dried in an incubator at ;@A., fi!ed in 93 acetic acid and stained with protein

stain. 0recipitin bands as lines result at the regions where the molecules have separated by electrophoresis. This techni$ue is used full for identifying the antigens in serum and to test purity of antigens. Thus it is used to identify bone marrow cancer and to study the gamma globulin in degenerative disease of nerve fibers Beferences -. Biophysics Principles and techni/ues By M.A Subramaniyan 0age 5umbers 9<2" 99> 0. Principles and "echni/ues of Biochemistry and Molecular Biology By 1eith ,ilson and 2ohn 3al4er C 0age 5umber 771 " 7>7