1. ELECTROPHORETIC METHODS

2. Basic principles of electrophoresis 1. It is the process of moving charged biomolecules in solution by applying an electrical field across the mixture. 2. Biomolecules moved with a speed dependent on their charge, shape, and size and separation occures on the basis of molecular size. Electrophoresis is used: for analysis and purification of very large molecules (proteins, nucleic acids) for analysis of simpler charged molecules (sugars, amino acids, peptides, nucleotides, and simpler ions). 3. When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. 1. 2. 3. 4.Factors influenced electrophoresis mobility: net charge of the molecule size and shape concentration of the molecule in solution 4. Electrophoresis is carried out by applying a thin layer Aqueous protein solution is immobilized in a solid hydrophilic support. Solid matrix with pores which are used: paper starch cellulose acetate polyacrylamide agar/agarose Molecules in the sample move through porous matrix at different velocity. 5. Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500). Most common stabilizing media are polyacrylamide or agarose gels. 6. Buffers Function of buffer 1. carries the applied current 2. established the pH 3. determine the electric charge on the solute High ionic strength of buffer produce sharper band produce more heat Commonly used buffer Barbital buffer & Tris-EDTA for protein Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8) 7. Gel electrophoresis Gel is a colloid in a solid form (99% is water). Gel material acts as a "molecular sieve. During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied. 8. Support media Agarose and polyacrylamide gels are across-linked, spongelike structure It is important that the support media is electrically neutral. Presence of charge group may cause: -Migration retardation -The flow of water toward one or the other electrode so called Electroendosmosis (EEO), which decrease resolution of the separation 9. Agarose highly purified polysaccharide derived from agar (extracted from seeweed), long sugar polymers held together by hydrogen and hydrophobic bonds. Acrylamide (CH2=CH-CONH2) Polyacrylamide gel structure held together by covalent cross-links 10. Agarose gels For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4%OH CH2OH O OOHO OOH OO 11. Polyacrylamide gels CH2=CHCONH2 Acrylamide+CH2(NHCOHC=CH2)2 N,N,N,N-methylenebisacrylamideFree radical catalyst -CH2-CH-CH2-CH-CH2-CHCO CO CO NH NH2 NH n CH2 CH2 NH NH2 NH CO CO CO -CH2-CH-CH2-CH-CH2-CHn 12. Capillary electrophoresis Capillaries are typically of 50 m inner diameter and 0.5 to 1 m in length. Due to electroosmotic flow, all sample components migrate towards the negative electrode. The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection.