Abstract
BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs pulse-width-modulated (PWM) voltage that applies a higher average voltage to a larger protein species. BlotMan can be controlled not only by its custom-made interface but also by a smart phone via Bluetooth technology. In this study, we examined effects of PWM signals (50%, 60%, and 80% duty cycle) on transfer efficiency and signal intensity in comparison to a constant voltage signal (100% duty cycle). The result revealed that in response to the same average voltage of 150 V, a lower duty cycle with a higher maximum voltage increased transfer efficiency as well as sharpness of transferred proteins. We validated BlotMan’s capability using a chondrosarcoma cell line (SW1353 cells) and a breast cancer cell line (MDA-MB231 cells) in response to antitumor chemical agents. BlotMan successfully generated 5 membranes from a single gel and detected 5 protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2), phosphorylated eIF2, lamin B, and actin. Collectively, we demonstrated herein that BlotMan reduces an amount of protein samples by generating multiple membranes from a single gel and improving signal intensity with PWM voltage signals.
Highlights
Western blotting is the most widely used technique to determine the relative amount of proteins [1] [2]
We addressed a question: Does a PWM voltage signal (
This study using BlotMan demonstrates that PWM voltage signals in a vertical gel increase protein mobility as well as signal intensity
Summary
Western blotting is the most widely used technique to determine the relative amount of proteins [1] [2]. It has been well established for over 30 years, the procedure remains highly labor-and-resource intensive. Capillary electrophoresis presents a new procedure, it may induce potential problems such as capillary clogging, air bubble formation, sensitivity to temperature fluctuations, inconsistency in polymer quality, and background noise in antibody reactions [5]. BlotMan is able to generate three membranes from a single gel, and it contributes to significant reduction in labor and resources
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