ARTGEL: A temperature-regulated gel electrophoresis platform for quantitative studies of reversible association

Abstract

Title: ARTGEL: A temperature-regulated gel electrophoresis platform for quantitative studies of reversible association Abstract: Here we present ARTGEL, an actively regulated-temperature gel electrophoresis platform designed for quantitative studies of temperature-sensitive reversible biomolecular association. \added{Existing literature describes electrophoretic mobility shift assays in which biomolecular reactions are suppressed during electrophoresis through dilution, the use of competitors, or reduced temperature. These approaches can alter temperature-sensitive equilibria and may cause the gel to report a state different from that of the original bulk solution. Rather than attempting to quench reactions, ARTGEL enables electrophoresis to be performed at the same temperature as complementary bulk measurements, allowing reversible association to be quantified directly in the gel and compared with matched measurements in solution.} The ARTGEL platform integrates feedback-controlled thermoelectric regulation of gel temperature, a large, heated, and continuously circulated buffer reservoir to help maintain ionic strength, and automated electrode-wiping to maintain stable voltage across the gel during long-duration electrophoresis. \added{During electrophoresis, ARTGEL maintained the gel temperature at a 30 °C setpoint with a temporal variation of 0.03 °C and a spatial range of 0.6 °C. Migration-velocity measurements provided indirect evidence that the voltage drop across the gel changed by no more than approximately $5\%$ over the 24-h experiment.} Using DNA origami assemblies, we further show that ARTGEL preserves distinct temperature-dependent association states, resolves reaction-dependent distortions of migrating bands, and supports extraction of in-gel kinetic and thermodynamic parameters from reaction–diffusion–advection modeling. Authors (w/ORCID): Rupam Saha - 0009-0000-9567-3010 Seth Fraden* - 0000-0002-2420-9939 Funding: This work is supported by the Brandeis University Materials Research Science and Engineering Center, which is funded by the National Science Foundation under award number DMR-2011846. Description: This repository contains the files required to reproduce and operate the instrument ARTGEL. A detailed description of the instrument can be found in the publication: ARTGEL a temperature-regulated agarose gel electrophoresis platform for quantitative studies of reversible association in gels (https://arxiv.org/abs/2606.09597). The repository is organized as follows: Bill_of_Materials/ BOM.xlsx – Bill of materials listing the components required to build the ARTGEL instrument. Firmware/ TEC_control.ino – Arduino firmware for regulating the thermoelectric (Peltier) module used to control the gel temperature. Buffer_temp_control.ino – Arduino firmware for regulating the immersion heater used to control the buffer reservoir temperature. Software/ user_interface.mlapp – MATLAB graphical user interface (GUI) for operating the instrument. The GUI enables users to monitor and control the gel and buffer temperatures, configure proportional–integral (PI) controller parameters, set the electrophoresis voltage and experiment duration, and record experimental data. Hardware/ plug.stl – 3D-printable model of the well plugs used to block gel wells during long-duration electrophoresis, preventing electrochemical deposits from entering the wells. Documentation/ SI_material.pdf – Supplementary information accompanying the HardwareX manuscript, including additional experimental methods, calibration procedures, controller tuning, and supporting figures. Data/ Scanned gel images/ The raw laser-scanned images of the gels. The file name starts with the reference to the figure in the manuscript or supplementary.pdf. Use ImageJ to open the .gel files. Supplementary/ Movie_S1.mp4 – Supplementary video demonstrating the operation of the ARTGEL instrument during electrophoresis, including buffer circulation, automated electrode wiping, and real-time temperature regulation. Movie_S1_thumbnail.png – Thumbnail image corresponding to Supplementary Movie S1. Analysis_code/ o plot_current_vs_time/ plot_current_time.m : MATLAB code to plot electrophoresis current as a function of time. o plot_intensity_profiles/normalize.m : MATLAB code to plot background-subtracted and normalized intensity profiles, obtained from IMAGEJ/FIJI. For building instructions of ARTGEL, please follow the section 5 of the main manuscript.