Institution: | 1. T-mac Co. Ltd., Daejeon, Korea
Both authors contributed equally to this work.;2. Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Korea
Both authors contributed equally to this work.;3. Department of Biological Sciences and Biotechnology, Hannam University, Daejeon, Korea;4. Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Korea;5. T-mac Co. Ltd., Daejeon, Korea |
Abstract: | 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. |