electrophoresis tech note 1156 Acrylamide Polymerization — A Practical Approach Paul Menter, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Polyacrylamide Gel Polymerization Hercules, CA 94547 USA AcrylamideBis Polyacrylamide Introduction The unparalleled resolution and flexibility possible with CH2 CH + CH2 CH CH2 CH CH2 CH CH2 CH polyacrylamide gel electrophoresis (PAGE) has led to its CO CO CO CO CO widespread use for the separation of proteins and nucleic NH2 NH NH2 NH2 NH acids. Gel porosity can be varied over a wide range to meet CH2 CH2 specific separation requirements. Electrophoresis gels and NH NH NH NH buffers can be chosen to provide separation on the basis of CO 2 2 CO CO C O charge, size, or a combination of charge and size. CH2 CH CH2 CH CH2 CH CH2 CH The key to mastering this powerful technique lies in the polymerization process itself. By understanding the important Purity of Gel-Forming Reagents parameters, and following a few simple guidelines, the novice Acrylamide can become proficient and the experienced user can optimize Gel-forming reagents include the monomers, acrylamide and bis, separations even further. as well as the initiators, usually ammonium persulfate and TEMED or, occasionally, riboflavin and TEMED. On a molar This bulletin takes a practical approach to the preparation of basis, acrylamide is by far the most abundant component in the polyacrylamide gels. Its purpose is to provide the information monomer solution. As a result, acrylamide may be the primary required to achieve reproducible, controllable polymerization. source of interfering contaminants (Dirksen and Chrambach For those users interested only in the “bare essentials,” the 1972). Poor-quality acrylamide contains significant amounts of Polymerization Protocols can be used as a laboratory guide. the following contaminants: Mechanism of Polymerization 1. Acrylic acid — Acrylic acid is the deamidation product of Polyacrylamide gels are formed by copolymerization of acrylamide. Acrylic acid will copolymerize with acrylamide acrylamide and bis-acrylamide (“bis,” N,N'-methylene-bis- and bis, thereby conferring ion exchange properties on the acrylamide). The reaction is a vinyl addition polymerization resulting gel. This can lead to local pH changes in the gel and initiated by a free radical-generating system (Chrambach 1985). cause artifacts such as aberrant relative mobility, precipitation of Polymerization is initiated by ammonium persulfate and TEMED some proteins and nucleic acids, streaking or smearing of (tetramethylethylenediamine): TEMED accelerates the rate of bands, and run-to-run irreproducibility. In acrylamide, acrylic acid formation of free radicals from persulfate and these in turn should be below 0.001% (w/w). This is determined by direct catalyze polymerization. The persulfate free radicals convert titration, and supported by both conductivity and acrylamide monomers to free radicals which react with pH measurement. unactivated monomers to begin the polymerization chain reaction (Shi and Jackowski 1998). The elongating polymer 2. Linear polyacrylamide — Contaminants with catalytic properties chains are randomly crosslinked by bis, resulting in a gel with may cause what appears to be autopolymerization during the a characteristic porosity which depends on the polymerization production, processing, or storage of marginally pure acrylamide. conditions and monomer concentrations. This results in the presence of linear polyacrylamide in the dry monomer. Linear polyacrylamide will affect polymerization, since it Riboflavin (or riboflavin-5'-phosphate) may also be used as a serves as a nucleus for polymerization. The most important effect source of free radicals, often in combination with TEMED and is the loss of reproducibility in gel porosity and relative mobilities ammonium persulfate. In the presence of light and oxygen, of proteins and nucleic acids. Linear polyacrylamide is detected riboflavin is converted to its leuco form, which is active in as water or alcohol insolubles and should be <0.005% (w/w). initiating polymerization. This is usually referred to as photochemical polymerization. 3. Ionic contaminants — Ionic contaminants can include both Contaminants in Buffers inhibitors and accelerators of polymerization. Aside from Contaminants in buffer reagents (Tris, borate, acetate, acrylic acid, the most notable ionic contaminants are metals glycine, etc.), gel additives (SDS, urea, etc.), and laboratory such as copper, which can inhibit gel polymerization. water can have a profound effect on polymerization. The Metals can also poison enzymes, alter the relative mobility most common contaminants of these reagents are metals, of metal binding proteins such as calmodulin, and inhibit non-buffer ions, and breakdown products. The most frequent digestion of electrophoretically purified nucleic acids by effect of these contaminants is to inhibit polymerization. When restriction and modification enzymes. Ionic contaminants polymerization is partially inhibited, the resulting gel will have are detected indirectly by their effects on chemical and greater porosity than intended, and molecules will have photochemical polymerization, and by the conductivity of greater mobilities. Furthermore, control over polymerization monomer solutions. reproducibility is compromised. bis-Acrylamide Initiator Type and Concentration Bis is present in much smaller quantities than acrylamide Initiators are the effectors of polymerization. Of course, the rate in monomer solutions. However, improperly purified bis of polymerization depends on the concentration of initiators, contains some of the same contaminants as acrylamide. but more importantly, the properties of the resulting gel also These include products of autopolymerization and ionic depend on the concentration of initiators. Increasing the contaminants, which have the same deleterious effects, and concentration of initiators (e.g., ammonium persulfate and can be detected in the same ways, as the corresponding TEMED) results in a decrease in the average polymer chain acrylamide contaminants. length, an increase in gel turbidity, and a decrease in gel Initiators elasticity. In extreme cases, excess initiator can produce a gel Chemical polymerization is initiated by ammonium persulfate, solution that does not appear to polymerize at all. This is due while photochemical polymerization is initiated by riboflavin to the formation of polymer chains so short that visible (or riboflavin-5'-phosphate), or by a combination of riboflavin gelation does not take place and the polymer stays in solution. and ammonium persulfate. Initiation and polymerization are The only indication that a reaction has taken place is an catalyzed by TEMED. Because polymerization is initiated by increase in viscosity. the generation of free radicals from persulfate or riboflavin, Excess ammonium persulfate and TEMED have other it is not surprising that these compounds are reactive, and effects, including oxidation of sample proteins (especially prone to oxidation or decomposition. The contaminants of sulfhydryl-containing compounds) and changes in buffer pH. the initiators tend to be the products of their own breakdown Excess TEMED can increase buffer pH, react with proteins as well as other contaminating compounds. (Dirksen and Chrambach 1972; Chrambach et al. 1976), TEMED is subject to oxidation, which causes the gradual and alter the banding pattern (Gelfi and Righetti 1981a). loss of catalytic activity. This process is greatly accelerated Ammonium persulfate acts as a buffer between pH 8 and 9. by contaminating oxidizing agents. TEMED that contains Potassium persulfate is recommended instead of ammonium oxidation products is characterized by a yellow color. The persulfate in weakly buffered basic systems (~pH 9). practical consequences of the oxidative process are the Excess riboflavin may cause the oxidation of some requirement for greater amounts of TEMED to achieve compounds, especially sulfhydryl-containing compounds adequate polymerization, and a gradual loss of TEMED (Dirksen and Chrambach 1972), and can denature proteins reactivity with time. TEMED is also very hygroscopic and will (Righetti et al. 1981). gradually accumulate water, which will accelerate oxidative Reducing the concentration of initiators results in longer decomposition. TEMED with maximum activity and shelf life polymer chain lengths, lower turbidity, and greater elasticity. is obtained by redistillation immediately prior to bottling, These are desirable properties. However, lower initiator resulting in a product that is clear, water free, and greater concentrations also mean slower polymerization. If polymeri- than 99% pure (14.4 M). zation is too slow, oxygen will begin to enter the monomer Ammonium persulfate is also very hygroscopic. This property solution and inhibit polymerization, resulting in gels which are is particularly important, since ammonium persulfate begins too porous and mechanically weak. Inhibition will be especially to break down almost immediately when dissolved in water. pronounced at surfaces exposed to air, or at the surfaces Therefore, the accumulation of water in ammonium persulfate of combs and spacers, which appear to trap air at their results in a rapid loss of reactivity. This is why ammonium surfaces. The remaining unpolymerized monomer can react persulfate solutions should be prepared fresh daily. Persulfate with alpha amino, sulfhydryl, and phenolic hydroxyl groups of is consumed in the polymerization reaction.