Abstract 5484: Expanding the role of western blot imaging for oncology research: novel applications for in-cell western assays and single cell analysis using the ChemiDoc Go imaging system

Abstract Western blotting is widely used to detect cellular proteins and nucleic acids in cell lysates; however, its application for analyzing intact cells remains largely unexplored. Here, we evaluate compatibility of Bio-Rad's ChemiDocTM Go Imaging System with chemiluminescence and multi-channel fluorescence detection for in-cell western immunofluorescence assays performed on 96-well plates and microscopy slides. Emphasis was placed on use of the StarBright Blue 700-conjugated secondary antibody due to its strong, stable fluorescence, wide dynamic range, and compatibility with multiplexed assays. In 96-well assays, HEK293 cells were stained with anti-mortalin primary antibodies, followed by StarBright Blue 700-conjugated secondary antibodies. These exhibited a three-fold signal increase compared to control samples lacking the primary antibody. This scalable and high-throughput platform offers a valuable tool for cancer research, particularly for evaluating protein expression in cancer cell lines, facilitating drug discovery, and assessing therapeutic efficacy across diverse cancer models. On microscope slides, HEK293 cells were stained with anti-mortalin primary and StarBright Blue 700 secondary antibodies further demonstrating the imager’s strong compatibility with immunofluorescence on fixed, intact single cells. This capability enables simultaneous detection of multiple biomarkers in cancer-related pathways, supporting target validation and pathway analysis in cancer biology. Additionally, the imager was used to validate T cell staining with CD3-labeled fluorescent antibodies prior to multiplexed flow cytometry. This step simplifies the confirmation of T cell staining before performing more complex flow cytometry assays, with direct applications in immuno-oncology. These include monitoring immune cell activation and characterizing tumor-infiltrating lymphocytes (TILs), both critical for developing immune-based cancer therapies. Potential oncology research applications of these novel techniques may include: Quantification of protein expression changes following drug treatments or genetic modifications Evaluation of protein-protein interactions relevant to cancer signaling pathways High-throughput screening of compound libraries Provide insights into the analysis of protein localization within entire cells, aiding in biomarker identification and therapeutic targeting efforts. Together, these findings highlight novel applications of western blot imaging technology as a fast, scalable, and sensitive platform for multiplexed protein analysis, supporting cancer research and therapeutic development. Citation Format: Michael Kirmiz, Deanna Woo, Nikolas Chmiel, Kenneth Oh, Angelica P. Olcott. Expanding the role of western blot imaging for oncology research: novel applications for in-cell western assays and single cell analysis using the ChemiDoc Go imaging system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5484.