A Novel Application for Multiple Form Protein Separation by the Conjugation of Disc Electrophoresis and Isoelectric Focusing: Re-evaluating Cylindrical Disc-PAGE Tube System for Isoelectric Focusing and Potential Outcomes for Future Usage of this App

Aim: Various forms of isoelectric focusing, those involving a single glass column and stabilized by the presence of a sucrose gradient, are preferably used and are the most common pre-application step in 2D electrophoresis. However, the cylindrical disc - PAGE tube system is an exclusive method and has never been evaluated for isoelectric purposes. The goal here was to try a modified application for the separation of some multiple isoform proteins via the multiple tube disc gel system. And to establish isoform separation as a priority and evaluate the implications of this approach for future use. Methods: Essentially, basic features of disc and isoelectric focusing were integrated. Although conventional and therefore the essential component of disc electrophoresis requires three-layers of gel (sample, stacking and separation), only one form gel layer (i.e. separation) was configured, as a first attempt. As a second attempt, the homogenate and ampholite, were deliberately included in the unpolymerised gel medium as if they were gel preparation factors. Then, the separation of the isoforms proteins, which were thought to be homogenously distributed in the presence of ampholite, was achieved. Results: The observation of isoforms of proteins revealed the presence of a distinct pattern of protein bands in their respective isoionic areas. From an analytical perspective, this form of application exhibited very clear and resolved banding patterns. The results obtained regarding the isoforms of hemoglobin proteins are both surprising and informative for us. Conclusions: The banding pattern observed in present approach clearly showed multiple and distinct isoform character profiles. Therefore, this new approach is a clear reflection of the appearance and distinctive power of this novel application. It can be used for in the first dimension of conventional 2D electrophoresis for analytical purposes of multiple proteins separation, when followed vertically.