Improved Separation in Horizontal Protein SDS-PAGE with Double-Deck Flat Electrodes and a Field Inversion Gel Electrophoresis Module.

The horizontal flatbed electrophoresis method is employed to separate protein samples, providing greater flexibility for various electrophoretic applications and easier sample loading compared to its vertical counterpart. In the currently available equipment setup, cathode and anode electrodes are positioned on top of a gel at each end. Since an electric field enters the gel from the top, its strength gradually weakens from the top to the bottom of the gel. When examining the interior of gels following electrophoretic separation, the uneven electric field causes the protein bands to lie down forward in the direction of migration, leading to an increase in bandwidth. This issue has remained unaddressed for several decades. To address this problem, new clamp-shaped and double-deck electrodes were developed to apply an electric field simultaneously from both the top and bottom of the gel. Both of these new electrodes facilitated the formation of perpendicular protein band shapes and enhanced resolution at a comparable level. Due to their ease of use, double-deck electrodes are recommended. By combining these new electrodes with the field inversion gel electrophoresis (FIGE) technique, the protein bands could be focused and aligned nearly vertically, resulting in the highest level of electrophoretic resolution. Our electrodes are compatible with polyacrylamide gels of varying sizes, buffer systems, and sample well formats. They can be easily manufactured and seamlessly integrated into existing laboratory instruments for practical use.

Spreader-CDR system for efficient, reproducible, and frugal western blot.

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.

Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.

Western blot is a principal technique for the detection of specific proteins in various biology disciplines. The antibody incubation is an imperative step in western blot. Antibody incubation by mild shaking on a rocker is generally used to facilitate mixing of antibodies. However, mild shaking is an inefficient process to remove antibody depletion layer on blotting membrane and requires hours of incubation time to achieve antibody binding to target proteins. We propose an alternative method of cyclic draining and replenishing (CDR) incubation of antibody solution using a rotational incubation chamber. The study demonstrated that rotational CDR incubation could shorten antibody incubation time with enhanced sensitivity. Moreover, rotational CDR incubation could achieve a stronger antibody binding with lower antibody concentration when compared with batch incubation. In addition, rotational CDR incubation significantly improved the detection of low abundance proteins. This simple modification in western blot procedure makes it rapid, sensitive, and cost-efficient.