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bioRxiv · 10.64898/2026.06.03.729869

Development of a Staining-based Electrophoretic Mobility Shift Assay for Analyzing Pbx1, DNA and HoxA9 Interactions

Rai, K.·Olaosebikan, I. A.·Norouzi, P.·Pettipas, G.·Dadum, A. G.·Short, M.·Courtney, K. C.·Karatas Bristow, H.
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Abstract

Transcription factors (TFs) are master regulators of gene expression and control a wide range of cellular functions including embryonic development, signaling pathways, immune response, and differentiation. A tight regulation of gene expression is crucial during all stages of life and changes in the TF function can lead to developmental abnormalities, diseases such as cancer, or resistance to treatment. Therefore, TFs are a promising class of drug targets, and the techniques that would contribute to the development of TF modulators are critical. Electrophoretic Mobility Shift Assay (EMSA) has been a primary tool to verify protein-DNA interactions, where a fluorescent, biotin, or isotope end-labelled DNA probe is used to quantify binding. Such labeling techniques, however, can be costly, time consuming, possess safety hazard risks and require capital equipment for imaging. Here, we optimized a label-free, staining-based EMSA to characterize a potential drug target, homeodomain (HD) TF Pre-B-cell leukemia homeobox-1 (Pbx1) and its binding partner Homeobox A9 (HoxA9). Staining the polyacrylamide EMSA gel with a DNA intercalating green cyanine dye - SYBR safe - allowed the visualization of Pbx1 homeodomain interactions with DNA at nanomolar concentrations and enabled quantitative determination of protein-DNA apparent binding affinity in the sub-micromolar range. Furthermore, a ternary complex of homeodomains of Pbx1 with HoxA9 and the DNA was also visible in the assay. We have shown that the staining-based EMSA can be used to evaluate inhibitors of Pbx1 that block the interaction with DNA. We have further validated the data from our assay with fluorophore labeling-based EMSA. Overall, using HD transcription factors Pbx1 and HoxA9 as a model, we have optimized a reliable and cost-effective staining-based EMSA that enables the high-sensitivity visualization and quantitative evaluation of transcription factor-DNA complexes without the need for end-labeled DNA probes. The streamlined workflow could be readily adapted to other DNA-binding proteins to study their interactions with the DNA, inhibitors, and other proteins.

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Rai, K., Olaosebikan, I. A., Norouzi, P., Pettipas, G., Dadum, A. G., Short, M., Courtney, K. C., Karatas Bristow, H.. 2026-06-07. Development of a Staining-based Electrophoretic Mobility Shift Assay for Analyzing Pbx1, DNA and HoxA9 Interactions. https://doi.org/10.64898/2026.06.03.729869

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