Chapter 10 Protein Electrophoresis10.1 Introdution10.1.1 Principles of electrophoresisElectrophoresis is the process of moving charged molecules in solution by applying an electric field across the mixture (Fig 1). Because molecules in an electric field move with a speed dependent on their charge, shape, and size, electrophoresis has been extensively developed for molecular separations.As an analytical tool, electrophoresis is simple and relatively rapid. It is used chiefly for analysis and purification of very large molecules such as proteins and nucleic acids. Highly sensitive detection methods have been developed to monitor and analyze electrophoretic separations.Fig 1. Basic arrangement for electrophoresis.Electrophoresis of macromolecules （高分子）is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix（多孔基体）. Under the influence of an applied voltage（电压）, different species of molecules in the sample move through the matrix at different velocities（速度）. At the end of the separation, the different species are detected as bands （条带）at different positions in the matrix. A matrix is required because the electric current passing through the electrophoresis solution generates heat, which causes diffusion and convective （扩散与对流）mixing of the bands in the absence of a stabilizing medium.The matrix can be composed of a number of different materials, including paper, cellulose acetate, or gels made of polyacrylamide（聚丙烯酰胺）, agarose, or starch（淀粉）. 10.1.2 MatrixPolyacrylamide and agarose gels (Fig 2) are the most common matrix used in research laboratories. In these gels, the matrix also acts as a size-selective sieve （筛）in the separation. The size of the pores of these gels is similar to the sizes of many proteins and nucleic acids. As molecules are forced through the gel by the applied voltage, larger molecules are retarded （阻滞）by the gel more than are smaller molecules. At the end of the run, the separated molecules can be detected in position in the gel by staining（染色后在位检测）, quantified （定量）by scanning with a densitometer（光密度计）, and the gel can be dried for permanent storage. Fig 2. Chemical structure of acrylamide and agaroseBecause the pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins, and protein complexes. Polyacrylamide is the most common matrix for separating proteins. Nucleic acids are separated on either polyacrylamide or agarose gels, depending on the sizes of molecules to be analyzed.Whichever matrix is selected, it is important that it be electrically neutral（电中性）. Charged matrices（matrix的复数） may interact chromatographically with molecules and retard migration（迁移）. The presence of fixed charged groups on the matrix will also cause the flow of water toward one or the other electrode（电极）, usually the cathode（负极）. This phenomenon, called electroendosmosis(EEO，电内渗), usually decreases the resolution （分辨率）of the separation（especially exist in agarose gel）.1、 Agarose gelsAgarose is a highly purified polysaccharide derived from agar. Agarose can be used for separation of large proteins or DNA. Agarose is normally purchased as a dry powder. It dissolves when the suspended powder is heated to near boiling and it remains liquid until the temperature drops to about 40 C, when it gels （胶凝）or “sets.” There are specific types of agarose that have melting and gelling temperatures considerably lower than those of standard agarose. These properties improve the recovery of material from a gel after separation for subsequent enzymatic treatments of the separated material. The pore size and sieving characteristics of a gel are determined by adjusting the concentration of agarose in the gel. The higher the concentration, the smaller the pore size. Working concentrations are normally in the range of 0.44% w/v.2、 Polyacrylamide gelsPolyacrylamide gels are physically tougher than agarose gels. Crosslinked polyacrylamide matrix are formed from the polymerization of acrylamide（Acr） monomer in the presence of smaller amounts of N,N-methylene-bis-acrylamide (“Bis” for short). Acrylamide monomer is polymerized in a head-to-tail fashion into long chains, and occasionally a Bis molecule is built into the growing chain, thus introducing a second site for chain extension. Proceeding in this way, a crosslinked matrix is formed (Fig. 3). Fig 3. Polyacrylamide matrixThe polymerization of acrylamide is an example of free-radical catalysis, and is initiated by the addition of ammonium persulfate（APS） and the base N,N,N,N- tetramethylenediamine (TEMED). TEMED catalyzes the decomposition of the persulfate ion to give a free radical (i.e., a molecule with an unpaired electron): (1)If this free radical is represented as R (where the dot represents an unpaired electron) and M as an acrylamide monomer molecule, then the polymerization can be represented as f