Abstract

Protein detection is a cornerstone of life science research. Here, we present Cell Lysate Fluorescence Anisotropy (CFAST), a versatile and accessible alternative cellular protein detection method. CFAST measures cellular protein levels through changes in fluorescence anisotropy of a long lifetime triangulenium dye-labeled nanobody probe. The minimal steps of centrifugation and nuclease incubation remove unwanted cellular components. We demonstrate that CFAST robustly detects cellular PARP1, MK2, and GFP protein levels. Coupling small molecule incubation and thermal shift with CFAST (thermal shift-CFAST) enables detection of cellular protein-small molecule interactions through quantification of thermally stabilized target protein. Thermal shift-CFAST successfully detects cellular PARP1 and MK2 inhibitor binding in a dose-dependent manner. High-throughput thermal shift-CFAST screening identified a small molecule binder of undruggable transcription factor SOX2. This screening method also enabled development of a bifunctional PROTAC that simultaneously degrades membrane-bound oncogene EGFR and inhibits immune modulating target HRH1. CFAST is rapid, taking a minimum of 2–3 hours to execute. It is also straightforward, utilizing common lab equipment and reasonably priced reagents. Our work suggests that CFAST is a viable alternative method to enhance protein detection as well as drug and probe discovery that is scalable, fast, and economical.

Title

Versatile Detection of Cellular Protein via Fluorescence Anisotropy

Authors

Qing Tang, Yuan-Ping Wei, Ricky Ruiqi Ma, Na Wei, Tammy Zihan Zhou, Hilary Kung-Yu Ho, Zafar Iqbal Bhat, Iqra Ishrat, Xiaoyu Li, Judy Wai Ping Yam, Yung Hou Wong, Qiankun Wang, Justin L. Tan

Journal Information

Advanced Science (2026)

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