SDS-PAGE and Western Blotting
Proteins can be separated according to their molecular sizes and charges, since these factors will determine the speed at which they will travel through a gel. The SDS-PAGE method involves the denaturation of proteins with the detergent sodium dodecyl sul
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32 SDS-PAGE and Western Blotting Abdulhamid A. Al-Tubuly 1. Introduction Proteins can be separated according to their molecular sizes and charges, since these factors will determine the speed at which they will travel through a gel. The SDS-PAGE method involves the denaturation of proteins with the detergent sodium dodecyl sulfate (SDS) and the use of an electric current to pull them through a polyacrylamide gel, a process termed polyacrylamide gel electrophoresis (PAGE). SDS binds strongly to proteins, with approximately one detergent molecule binding to two amino acids when SDS is present at 0.1% (1,2). When boiled with SDS, proteins gain a negative charge in proportion to their molecular size, and thus travel in the acrylamide gel according to their molecular sizes. The smaller the size of the running protein, the faster it travels through the pores of the gel (Fig. 1). The polyacrylamide gel is formed by polymerization of monomers of acrylamide with monomers of an appropriate crosslinking substance. The most commonly used crosslinking agent is N,Nv-methylene-bis-acrylamide (usually abbreviated bisacrylamide, or BIS). Gel polymerization is usually initiated with ammonium persulfate (APS), and the reaction is accelerated by addition of the catalyst N,N,Nv,Nv-tetramethylenediamine (TEMED). A threedimensional network is then formed by the crosslinking of randomly growing polyacrylamide chains. The concentrations of acrylamide and the crosslinking agent used will determine the length of the polymers and the extent of the crosslinking, which in turn will affect the physical properties of the gel, such as density, elasticity, fragility, and, most importantly, pore size (3,4). Careful consideration must, therefore, be given to the choice of acrylamide concentration for the optimal separation of different proteins. Gels with large pore size allow faster running of large size proteins, whereas gels with high density (i.e., From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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Fig. 1. Migration of different size proteins in a vertical flat slab gel. The smaller the molecular size of the protein, the smaller the charge conferred to it. Migration through the gel is determined by the pore size of the gel, with smaller molecules moving faster.
smaller pore size) would slow large proteins down, permitting better separation of smaller molecules (Table 1). The best way to analyze a novel protein with no prior information about its size range is to adopt a method of trial and error, in which gels of different concentrations are tested. The two main approaches for SDS treatment of proteins are the reducing and nonreducing methods. In the former approach, by adding a reducing thiol agent, such as urea, 2-mercaptoethanol, or dithiothreitol (DTT), to the sample buffer, the proteins will totally unfold and a separation according to their molecular weight is possible (see Note 1). In the latter (nonreduci
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