Best polymerization conditions for acrylamide gels are obtained at 25–30 °C and polymerization seems terminated after 20–30 min of reaction although residual monomers (10–30%) are detected after this time. The co-polymerization of acrylamide (AA) monomer/N,N'-Methylenebisacrylamide (Bis-AA) cross-linker initiated by ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) reactions, is most efficient at alkaline pH of the acrylamide solution. Thereby, acrylamide chains are created and cross-linked at a time. Due to the properties of the electrophoresis buffer, the gel polymerization is conducted at pH 10.00 making sure an efficient use of TEMED and APS as catalysts of the polymerization reaction, and concurrently, suppressing a competitive hydrolysis of the produced acrylamide polymer network. Polymer networks are three-dimensionally linked polymer chains. Otherwise, proteins could be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products that may result in multiple bands.
Additionally, the time of polymerization of a gel may directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression and dilatation of the gels and their pores if the incubation times for the reaction mixture (gel solution) used to prepare a gel are not optimized (cf. fig. Electropherogram, see sect. Reproducibility and recovery). In order to ensure maximum reproducibility in gel pore size and to obtain a fully polymerized and non-restrictive large pore gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature (RT) in a gel column located on the casting stand. The exothermic heat generated by the polymerization processes is dissipated constantly while the temperature may rise rapidly to over 75 °C in the first minutes, after which it falls slowly. After 69 hr, the gel has reached room temperature and is in its lowest energy state, as the basic chemical reactions and gelation are complete. Gelation means that the
solvent (water) gets immobilized within the polymer network by means of hydrogen bonds and also van der Waals forces. As a result, the prepared gel is homogeneous (in terms of homogeneous distribution of cross-links throughout the gel sample), inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins. Sieving effects due to gravity-induced compression of the gel can be excluded for the same reasons. Thus, in a medium without molecular sieving properties a high-resolution can be expected.
Before an electrophoretic run is started the prepared 4% T (total polymer content (T)), 2.67% C (cross-linker concentration (C)) gel is pre-run to equilibrate it. It is essentially non-sieving and optimal for electrophoresis of proteins greater than or equal to 200 ku. Proteins migrate in it more or less on the basis of their free mobility. For these reasons interactions of the gel with the biomolecules are negligibly low, and thus, the proteins separate cleanly and predictably at a polymerization time of 69 hr (cf. fig. Electropherogram). The separated metalloproteins including biomolecules ranging from approximately < 1 ku to greater than 30 ku (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides, metallothionein, phytochelatins) are not dissociated into apoproteins and metal cofactors.