Detection of the DNA binding of transcription factors in situ at the single-cell resolution in cultured cells by proximity ligation assay

Transcription factors (TFs) play a pivotal role in gene expression, and their DNA binding is the prerequisite to instigating gene transcription. Here, we present a protocol that exploits the proximity ligation assay technique to measure the DNA-binding activities of TFs in situ at the single-cell resolution. We describe steps for immunostaining with specific antibodies against double-stranded DNA and the TFs of interest, probe incubation, proximity ligation, and signal amplification. We then detail procedures for imaging and image analysis. For complete details on the use and execution of this protocol, please refer to Dai et al. (2015)1 and Xu et al. (2023).2 [Display omitted] •Adaptation of the proximity ligation assay to visualize protein-DNA interactions•In situ single-cell measurement under physiological conditions•A versatile protocol applicable to numerous DNA-binding proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Transcription factors (TFs) play a pivotal role in gene expression, and their DNA binding is the prerequisite to instigating gene transcription. Here, we present a protocol that exploits the proximity ligation assay technique to measure the DNA-binding activities of TFs in situ at the single-cell resolution. We describe steps for immunostaining with specific antibodies against double-stranded DNA and the TFs of interest, probe incubation, proximity ligation, and signal amplification. We then detail procedures for imaging and image analysis.