Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 Pharma Science Monitor 7(2),Apr-Jun 2016 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com REVIEW ON: ELECTROPHORESIS: METHOD FOR PROTEIN SEPARATION ShindeDipa V.*, JasminaSurati Department of Quality Assurance, Shree NaranjibhaiLalbhai Patel College of Pharmacy,Umrakh -394 345,Bardoli, Gujarat, India. ABSTRACT Electrophoresis is one of the widely used techniques in molecular biochemistry, microbiology, biomedical research. It is a type of protein separation method .It is one of the highly efficient techniques of analysis and sole method for separation of proteins for western blot, RNA studies etc. It is a both qualitative and quantitative analysis technique. Separation depend upon electrophoretic mobility.Electrophoresis technique are of various type like Moving boundary electrophoresis ,Zone electrophoresis ,Affinity electrophoresis ,Pulsed field electrophoresis ,Dielectrophoresis.this technique mainly used in antibiotic analysis,vaccine analysis DNA analysis and protein analysis as well as fingerprint analysis. KEYWORDS:Electrophoresis, Electrophoretic mobility,Zone Electrophoresis, Moving boundary Electrophoresis, Dielectricphoresis. INTRODUCTION Electrophoresis is a physical method of analysis based on the migration of electrically charged proteins, colloids, molecules or other particles dissolved or dispersed in an electrolyte solution in the direction of the electrode bearing the opposite polarity when an electric current is passed through it. Separations may be conducted in systems without support phases (such as free solution separation in capillary electrophoresis) or in stabilising media such as thin-later plates, filins or gels. The electrophoretic mobility is the rate of movement in metres per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second. For practical reasons it is given in squarecentimetres per volt second cm2 V-iS-I. The mobility is specific for a given electrolyte under precisely determined operational conditions. Depending on the method used, the electrophoretic mobility is either measured directly or compared with that of a reference substance. Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 193 Principle of Electrophoresis: The term Electrophoresis means Electro=electric field + Phoresis=migration. So as the name indicates, “Electrophoresis is a method of separation where in charged molecules migrate in differential speeds in an applied electric field.” The charged molecules under the influence of electric field migrate towards oppositely charged electrodes. Those molecules with +ve charge move towards cathode and -ve molecules move towards Anode. The migration is due to charge on the molecules and potential applied across the electrodes. The sample under test is placed at one end of the paper near one of electrodes. When electricity is applied, the molecules start moving to respective electrodes. But the movement is influenced by molecular weight of the molecule. So when a mixture isplaced on the electrophoresis paper or gel, different bands are seen along the paper after the process.This is due differentialrate of movement by molecules based on their weight. Those molecules with higher molecular weight move slower. While those with small weight move faster. Also the size of the molecule also influences the movement. The bigger size molecule experience more friction than smaller ones in motion. These molecules migrate at different speed and to different lengths based on their charge, mass and shape. Based on type of apparatus used in electrophoretic method divided in two category: 1) Zone electrophoresis a) Paper electrophoresis b) Gel electrophoresis c) Thin layer electrophoresis d) Cellulose acetate electrophoresis Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 194 2) Moving boundary electrophoresis a) Capillary electrophoresis b) Isoatchophoresis c) Isoelectric focussing d) Immuno electrophoresis. Also other type of electrophoresis is : a) Affinity electrophoresis b) Dielectrophoresis c) Electroblotting d) Plused field gel electrophoresis 1) ZONE ELECTROPHORESIS(4-5) : This method requires the use of small samples only.In zone electrophoresis, the sample is introduced as a narrow zone or spot in a column, slab or film of buffer. Migration of the components as narrow zones perrrrits their complete separation. Remixing of the separated zones by thermal convection is prevented by stabilising the electrolyte in a porous matrix such as powdered solid, or a fibrous material such as paper, foil or gel such as agar, starch or polyarcylamide. The rate of migration depends on four main factors viz. The mobility of the charged particle, the electro-endosmoticflow,the evaporation flow, and the strength of the field. It is necessary to operate under clearly defined experimental conditions and to use, wherever possible, reference substances. a) Paper electrophoresis : Paper electrophoresis is a techniques which employs a Whattman filter paper No.1 which is moistened by a buffer and then connected at two ends to two opposite charged electrodes. Then sample is applied on to one end and let for separation of components under electric field. After separation, the paper is dried and stained to get colored bands. These colored bands are recognized for the nature of sample by comparing with the standard. For a sample of serum, 5 bands of proteins can be separated by paper electrophoresis. Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 195 b) Gel electrophoresis (6) : Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles. Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 196 Type of gel gel electrophoresis are classified as 1) Agarose gel electrophoresis 2) Polyacrylamide gel electrophoresis 1) Agarose gel electrophoresis : Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix of agarose. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.[2] 2) Polyacrylamide gel electrophoresis : Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biologyand biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule. As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins,sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for instance many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS.[1] Dipa et al. / Pharma Science Monitor 7(2),Apr-Jun 2016, 192-203 Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 197 2 )MOVING BOUNDARY ELECTROPHORESIS(7) :This method is chiefly employed with substances of high relative molecular weight and low diffusibility. In the method a buffered solution of proteins in a U-shaped cell is subjected to an electric current which causes the proteins to form series of layers in order of decreasing mobility, which are